U.S. patent application number 10/469679 was filed with the patent office on 2006-03-23 for modified interferon alpha with reduced immunogenicity.
Invention is credited to Matthew Baker, Francis J. Carr, Graham Carter, Marian Hanlon, Tim Jones, John Watkins.
Application Number | 20060062761 10/469679 |
Document ID | / |
Family ID | 8176646 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060062761 |
Kind Code |
A1 |
Carr; Francis J. ; et
al. |
March 23, 2006 |
Modified interferon alpha with reduced immunogenicity
Abstract
The present invention relates to polypeptides to be administered
especially to humans and in particular for therapeutic use. The
polypeptides are modified polypeptides whereby the modification
results in a reduced propensity for the polypeptide to elicit an
immune response upon administration to the human subject. The
invention in particular to the modification of human interferon
alpha and specifically interferon alpha 2(INF.alpha.2) to result in
proteins that are substantially non-immunogenic or less immunogenic
than any non-modified counterpart when use in vivo.
Inventors: |
Carr; Francis J.;
(Aberdeenshire, GB) ; Carter; Graham;
(Abendeenshire, GB) ; Jones; Tim; (Cambridge,
GB) ; Baker; Matthew; (Cambridge, GB) ;
Watkins; John; (Cambridge, GB) ; Hanlon; Marian;
(Cambridge, GB) |
Correspondence
Address: |
OLSON & HIERL, LTD.
20 NORTH WACKER DRIVE
36TH FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
8176646 |
Appl. No.: |
10/469679 |
Filed: |
March 1, 2002 |
PCT Filed: |
March 1, 2002 |
PCT NO: |
PCT/EP02/02218 |
371 Date: |
September 2, 2003 |
Current U.S.
Class: |
424/85.7 ;
435/320.1; 435/325; 435/69.51; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 7/06 20130101; A61P
37/02 20180101; C07K 14/56 20130101; A61P 43/00 20180101; A61K
38/00 20130101 |
Class at
Publication: |
424/085.7 ;
530/351; 435/069.51; 435/320.1; 435/325; 536/023.5 |
International
Class: |
C07K 14/56 20060101
C07K014/56; C07H 21/04 20060101 C07H021/04; C12P 21/04 20060101
C12P021/04; A61K 38/21 20060101 A61K038/21 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 2001 |
EP |
01105088.7 |
Claims
1. A modified molecule having the biological activity of human
interferon alpha 2 (INF.alpha.2) and being substantially
non-immunogenic or less immunogenic than any non-modified molecule
having the same biological activity when used in vivo.
2. A molecule according to claim 1, wherein said loss of
immunogenicity is achieved by removing one or more T-cell epitopes
derived from the originally non-modified molecule and/or by
reduction in numbers of MHC allotypes able to bind peptides derived
from said molecule.
3. A molecule according to claim 2, wherein one T-cell epitope is
removed.
4-70. (canceled)
71. A protein that is homologous to human interferon alpha 2
(INF.alpha.2), the human interferon alpha 2 having an amino acid
sequence (SEQ ID NO: 1) that includes at least one T-cell epitope;
the protein having substantially the same amino acid sequence as
SEQ ID NO: 1, but including at least one less T-cell epitope;
wherein the protein has substantially the same biological activity
as human interferon alpha 2, but is less immunogenic than said
human interferon alpha 2 when both are exposed to the immune system
of the same species.
72. The protein of claim 71 wherein the amino acid sequence of the
protein includes one less T-cell epitope.
73. The protein of claim 71 wherein the amino acid sequence of the
protein differs from SEQ ID NO: 1 by one to nine amino acid
residues.
74. The protein of claim 71 wherein the amino acid sequence of the
protein has at least one less amino acid residue than SEQ ID NO:
1.
75. The protein of claim 71 wherein the amino acid sequence of the
protein has at least one more amino acid residue than SEQ ID NO:
1.
76. The protein of claim 71 wherein the amino acid sequence of the
protein has the same number of amino acid residues as SEQ ID NO:
1.
77. A pharmaceutical composition comprising a protein of claim 71
and a pharmaceutically acceptable carrier therefor.
78. A method of preparing a protein of claim 71, the method
comprising the steps of: (i) identifying one or more potential
T-cell epitopes within the amino acid sequence of human interferon
alpha 2 (SEQ ID NO: 1); (ii) selecting at least one sequence
variant of at least one potential T-cell epitope identified in step
(i) that eliminates or substantially reduces the MHC class II
binding activity of the potential T-cell epitope; wherein the amino
acid sequence of the selected variant differs from the amino acid
sequence of the T-cell epitope identified in step (i) by at least
one amino acid residue; (iii) preparing, by recombinant DNA
techniques, at least one protein that includes at least one variant
selected in step (ii); (iv) evaluating the biological activity and
immunogenicity of at least one protein prepared in step (iii); and
(v) selecting a protein evaluated in step (iv) that has
substantially the same biological activity as, but substantially
less immunogenicity than human interferon alpha 2.
79. The method of claim 78 wherein step (i) is carried out by
determining the MHC class II binding affinity of potential T-cell
epitope segments of human interferon alpha 2 using an in vitro
assay, an in silico technique, or a biological assay.
80. The method of claim 78 wherein step (i) is carried out by: (a)
selecting a region of the amino acid sequence of human interferon
alpha 2 (SEQ ID NO: 1); (b) sequentially sampling overlapping amino
acid residue segments of predetermined uniform size and including
at least three amino acid residues from the selected region; (c)
calculating the MHC class II molecule binding score for each of the
sampled segments by summing assigned values for each hydrophobic
amino acid residue side chain present in the sampled amino acid
residue segment; and (d) identifying at least one segment that is
suitable for modification based on the calculated MHC class II
binding score for that segment to reduce the overall MHC class II
binding score for the protein relative to the binding score for
human interferon alpha 2.
81. The method of claim 80 wherein step (c) is carried out by using
a Bohm scoring function modified to include a van der Waal's
ligand-protein energy repulsive term and a ligand conformational
energy term by: (1) selecting a model from a first database of MHC
class II molecule models; (2) selecting an allowed peptide backbone
from a second database of allowed peptide backbones for the MHC
class II molecule models in step (1); (3) identifying amino acid
residue side chains present in each sampled segment; (4)
determining the binding affinity value for all side chains present
in each sampled segment; and (5) repeating each of steps (1)
through (4) for each model in the first database and for each
backbone in the second database.
82. The method of claim 78 wherein step (ii) is carried out by
substitution, addition, or deletion of one to nine amino acid
residues from a potential T-cell epitope identified in step
(i).
83. A protein of claim 71 having an amino acid sequence that is
free from T-cell epitopes.
84. A protein having the following amino acid sequence (SEQ ID NO:
5):
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
STKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKX.sup.1X.sup.2QRX.su-
p.3TX4YLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE, wherein X.sup.0 is R
or K; X.sup.1 is Y, E, or Q; X.sup.2 is F or H; X.sup.3 is I or A;
and X.sup.4 is L or A; excluding proteins having amino acid
sequences in which, simultaneously, X.sup.1 is Y, X.sup.2 is F,
X.sup.3 is I, and X.sup.4 is L.
85. A protein having the following amino acid sequence (SEQ ID NO:
6):
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
STKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPX.sup.1X2KEDSX.sup.3X.sup.4AVRKX-
.sup.5X.sup.6QRX.sup.7TX.sup.8YLKEKKYSPCAWEVVRAEIMRSFSX.sup.9STNLQESLRSKE,
wherein X.sup.0 is R or K; X.sup.1 is L, S, or G; X.sup.2 is M, T,
S, or E; X.sup.3 is I, S, or Q; X.sup.4 is L or G; X.sup.5 is Y, E,
or Q; X.sup.6 is F or H; X.sup.7 is I or A; X.sup.8 is L or A; and
X.sup.9 is L or S; excluding proteins having amino acid sequences
in which, simultaneously, X.sup.1 is L; X.sup.2 is M; X.sup.3 is;
X.sup.4 is L; X.sup.5 is Y; X.sup.6 is F; X.sup.7 is I; X.sup.8 is
L; and X.sup.9 is L.
86. A protein having the following amino acid sequence (SEQ ID NO:
7):
CDLPQTHSLGSRRTLMLLAQMRX0ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQX1X2NX3X4S-
TKDSSAAX5DETLLDKX6X7TELX8QQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPC-
AWEVVRAEIMRSFSLSTNLQESLRSKE, wherein X.sup.0 is R or K; X.sup.1 is
1 or T; X.sup.2 is F, D, or A; X.sup.3 is L or A; X.sup.4 is F, D,
or E; X.sup.5 is W or H; X.sup.6 is F, D, or E; X.sup.7 is Y or S;
and X.sup.8 is Y, D, E, or N; excluding proteins having amino acid
sequences in which, simultaneously, X.sup.1 is I; X.sup.2 is F;
X.sup.3 is L; X.sup.4is F; X.sup.5 is W; X.sup.6 is F; X.sup.7 is
Y; and X.sup.8 is Y.
87. A protein having the following amino acid sequence (SEQ ID NO:
8):
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQX.sup-
.1FNLFSTKDSSAAWDETLLDKFX.sup.2TELX.sup.3QQLNDLEACVIQGVGVTETPLMKEDSILAVRKYF-
QRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE, wherein X.sup.0 is R or
K; X.sup.1 is I or T; X.sup.2 is Y or S and X.sup.3 is Y, D, E, or
N; excluding proteins having amino acid sequences in which,
simultaneously, X.sup.1 is I; X.sup.2 is Y; and X.sup.3 is Y.
88. A protein having the following amino acid sequence (SEQ ID NO:
9):
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISX.sup.1X.sup.2SCLKDRHDFGX.sup.3PQEEFGNQFQK-
AETIPX.sup.4LHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSIL-
AVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE, wherein X.sup.0
is R or K; X.sup.1 is L or P; X.sup.2 is F or S; X.sup.3 is F or E;
and X.sup.4 is V or A; excluding proteins having amino acid
sequences in which, simultaneously, X.sup.1 is L; X.sup.2 is F;
X.sup.3 is F; and X.sup.4 is V.
89. An isolated polypeptide selected from the group of polypeptides
set forth in FIG. 1.
90. An isolated polypeptide selected from the group of polypeptides
set forth in FIG. 7.
91. An isolated polynucleotide encoding a protein of claim 71.
92. An isolated polynucleotide encoding a protein of claim 84.
93. An isolated polynucleotide encoding a protein of claim 85.
94. An isolated polynucleotide encoding a protein of claim 86.
95. An isolated polynucleotide encoding a protein of claim 87.
96. An isolated polynucleotide encoding a protein of claim 88.
97. An isolated polynucleotide encoding a polypeptide of claim
89.
98. An isolated polynucleotide encoding a polypeptide of claim
90.
99. A plasmid comprising a polynucleotide of claim 91.
100. A plasmid comprising a polynucleotide of claim 92.
101. A plasmid comprising a polynucleotide of claim 93.
102. A plasmid comprising a polynucleotide of claim 94.
103. A plasmid comprising a polynucleotide of claim 95.
104. A plasmid comprising a polynucleotide of claim 96.
105. A plasmid comprising a polynucleotide of claim 97.
106. A plasmid comprising a polynucleotide of claim 98.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to polypeptides to be
administered especially to humans and in particular for therapeutic
use. The polypeptides are modified polypeptides whereby the
modification results in a reduced propensity for the polypeptide to
elicit an immune response upon administration to the human subject.
The invention in particular relates to the modification of human
interferon and specifically human interferon .alpha.2 (INF.alpha.2)
to result in INF.alpha.2 protein variants that are substantially
non-immunogenic or less immunogenic than any non-modified
counterpart when used in vivo. The invention relates furthermore to
T-cell epitope peptides derived from said non-modified protein by
means of which it is possible to create modified INF.alpha.2
variants with reduced immunogenicity.
BACKGROUND OF THE INVENTION
[0002] There are many instances whereby the efficacy of a
therapeutic protein is limited by an unwanted immune reaction to
the therapeutic protein. Several mouse monoclonal antibodies have
shown promise as therapies in a number of human disease settings
but in certain cases have failed due to the induction of
significant degrees of a human anti-murine antibody (HAMA) response
[Schroff, R. W. et al (1985) Cancer Res. 45: 879-885; Shawler, D.
L. et al (1985) J. Immunol. 135: 1530-1535]. For monoclonal
antibodies, a number of techniques have been developed in attempt
to reduce the HAMA response [WO 89/09622; EP 0239400; EP 0438310;
WO 91/06667]. These recombinant DNA approaches have generally
reduced the mouse genetic information in the final antibody
construct whilst increasing the human genetic information in the
final construct. Notwithstanding, the resultant "humanized"
antibodies have, in several cases, still elicited an immune
response in patients [Issacs J. D. (1990) Sem. Immunol. 2: 449,
456; Rebello, P. R. et al (1999) Transplantation 68:
1417-1420].
[0003] Antibodies are not the only class of polypeptide molecule
administered as a therapeutic agent against which an immune
response may be mounted. Even proteins of human origin and with the
same amino acid sequences as occur within humans can still induce
an immune response in humans. Notable examples include the
therapeutic use of granulocyte-macrophage colony stimulating factor
[Wadhwa, M. et al (1999) Clin. Cancer Res. 5: 1353-1361] and
INF.alpha.2 [Russo, D. et al (1996) Bri. J. Haem. 94: 300-305;
Stein, R. et al (1988) New Engl. J. Med. 318: 1409-1413] amongst
others.
[0004] A principal factor in the induction of an immune response is
the presence within the protein of peptides that can stimulate the
activity of T-cells via presentation on MHC class II molecules,
so-called "T-cell epitopes". Such potential T-cell epitopes are
commonly defined as any amino acid residue sequence with the
ability to bind to MHC Class II molecules. Such T-cell epitopes can
be measured to establish MHC binding. Implicitly, a "T-cell
epitope" means an epitope which when bound to MHC molecules can be
recognized by a T-cell receptor (TCR), and which can, at least in
principle, cause the activation of these T-cells by engaging a TCR
to promote a T-cell response. It is, however, usually understood
that certain peptides which are found to bind to MHC Class II
molecules may be retained in a protein sequence because such
peptides are recognized as "self" within the organism into which
the final protein is administered.
[0005] It is known, that certain of these T-cell epitope peptides
can be released during the degradation of peptides, polypeptides or
proteins within cells and subsequently be presented by molecules of
the major histocompatability complex (MHC) in order to trigger the
activation of T-cells. For peptides presented by MHC Class II, such
activation of T-cells can then give rise, for example, to an
antibody response by direct stimulation of B-cells to produce such
antibodies.
[0006] MHC Class II molecules are a group of highly polymorphic
proteins which play a central role in helper T-cell selection and
activation. The human leukocyte antigen group DR (HLA-DR) are the
predominant isotype of this group of proteins and are the major
focus of the present invention. However, isotypes HLA-DQ and HLA-DP
perform similar functions, hence the present invention is equally
applicable to these. The MHC class II DR molecule is made of an
alpha and a beta chain which insert at their C-termini through the
cell membrane. Each hetero-dimer possesses a ligand binding domain
which binds to peptides varying between 9 and 20 amino acids in
length, although the binding groove can accommodate a maximum of 11
amino acids. The ligand binding domain is comprised of amino acids
1 to 85 of the alpha chain, and amino acids 1 to 94 of the beta
chain. DQ molecules have recently been shown to have an homologous
structure and the DP family proteins are also expected to be very
similar. In humans approximately 70 different allotypes of the DR
isotype are known, for DQ there are 30 different allotypes and for
DP 47 different allotypes are known. Each individual bears two to
four DR alleles, two DQ and two DP alleles. The structure of a
number of DR molecules has been solved and such structures point to
an open-ended peptide binding groove with a number of hydrophobic
pockets which engage hydrophobic residues (pocket residues) of the
peptide [Brown et al Nature (1993) 364: 33; Stern et al (1994)
Nature 368: 215]. Polymorphism identifying the different allotypes
of class II molecule contributes to a wide diversity of different
binding surfaces for peptides within the peptide binding grove and
at the population level ensures maximal flexibility with regard to
the ability to recognize foreign proteins and mount an immune
response to pathogenic organisms. There is a considerable amount of
polymorphism within the ligand binding domain with distinct
"families" within different geographical populations and ethnic
groups. This polymorphism affects the binding characteristics of
the peptide binding domain, thus different "families" of DR
molecules will have specificities for peptides with different
sequence properties, although there may be some overlap. This
specificity determines recognition of Th-cell epitopes (Class II
T-cell response) which are ultimately responsible for driving the
antibody response to B-cell epitopes present on the same protein
from which the Th-cell epitope is derived. Thus, the immune
response to a protein in an individual is heavily influenced by
T-cell epitope recognition which is a function of the peptide
binding specificity of that individual's HLA-DR allotype.
Therefore, in order to identify T-cell epitopes within a protein or
peptide in the context of a global population, it is desirable to
consider the binding properties of as diverse a set of HLA-DR
allotypes as possible, thus covering as high a percentage of the
world population as possible.
[0007] An immune response to a therapeutic protein such as the
protein which is object of this invention, proceeds via the MHC
class II peptide presentation pathway. Here exogenous proteins are
engulfed and processed for presentation in association with MHC
class II molecules of the DR, DQ or DP type. MHC Class II molecules
are expressed by professional antigen presenting cells (APCs), such
as macrophages and dendritic cells amongst others. Engagement of a
MHC class II peptide complex by a cognate T-cell receptor on the
surface of the T-cell, together with the cross-binding of certain
other co-receptors such as the CD4 molecule, can induce an
activated state within the T-cell.
[0008] Activation leads to the release of cytokines further
activating other lymphocytes such as B cells to produce antibodies
or activating T killer cells as a full cellular immune response.
The ability of a peptide to bind a given MHC class II molecule for
presentation on the surface of an APC is dependent on a number of
factors most notably its primary sequence. This will influence both
its propensity for proteolytic cleavage and also its affinity for
binding within the peptide binding cleft of the MHC class II
molecule. The MHC class II/peptide complex on the APC surface
presents a binding face to a particular T-cell receptor (TCR) able
to recognize determinants provided both by exposed residues of the
peptide and the MHC class II molecule.
[0009] In the art there are procedures for identifying synthetic
peptides able to bind MHC class II molecules (e.g. WO98/52976 and
WO00/34317). Such peptides may not function as T-cell epitopes in
all situations, particularly, in vivo due to the processing
pathways or other phenomena. T-cell epitope identification is the
first step to epitope elimination. The identification and removal
of potential T-cell epitopes from proteins has been previously
disclosed. In the art methods have been provided to enable the
detection of T-cell epitopes usually by computational means
scanning for recognized sequence motifs in experimentally
determined T-cell epitopes or alternatively using computational
techniques to predict MHC class II-binding peptides and in
particular DR-binding peptides.
[0010] WO98/52976 and WO00/34317 teach computational threading
approaches to identifying polypeptide sequences with the potential
to bind a sub-set of human MHC class II DR allotypes. In these
teachings, predicted T-cell epitopes are removed by the use of
judicious amino acid substitution within the primary sequence of
the therapeutic antibody or non-antibody protein of both non-human
and human derivation.
[0011] Other techniques exploiting soluble complexes of recombinant
MHC molecules in combination with synthetic peptides and able to
bind to T-cell clones from peripheral blood samples from human or
experimental animal subjects have been used in the art [Kern, F. et
al (1998) Nature Medicine 4:975-978; Kwok, W. W. et al (2001)
TRENDS in Immunology 22: 583-588] and may also be exploited in an
epitope identification strategy.
[0012] As depicted above and as consequence thereof, it would be
desirable to identify and to remove or at least to reduce T-cell
epitopes from a given in principal therapeutically valuable but
originally immunogenic peptide, polypeptide or protein.
[0013] One of these therapeutically valuable molecules is
INF.alpha.2. The molecule is an important glycoprotein cytokine
expressed by activated macrophages. The protein has antiviral
activity and stimulates the production of at least two enzymes; a
protein kinase and an oligoadenylate synthetase, on binding to the
interferon alpha receptor in expressing cells. The mature
INF.alpha.2 protein is single polypeptide of 165 amino acids
produced by post-translational processing of a 188 amino acid
pre-cursor protein by cleavage of a 23 amino acid signal sequence
from the amino terminus. Several different subtypes of human
INF.alpha.2 are known showing minor differences between primary
amino acid sequences. Thus INF.alpha.2a and INF.alpha.2b differ in
only one residue at position 23 of the mature protein chain being
lysine in INF.alpha.2a and arginine in INF.alpha.2b. Whilst the
disclosures of the present invention are directed towards the
sequence of INF.alpha.2b, it can be seen that for all practical
purposes the sequence of INF.alpha.2a may be considered
interchangeably with the subject INF.alpha.2b subtype of the
present invention. The amino acid sequence of INF.alpha.2(a,b)
(depicted as one-letter code) is as follows: TABLE-US-00001
CDLPQTHSLGSRRTLMLLAQMR (R,K) ISLFSCLKDRHDFGFPQEEFG
NQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLND
LEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVR
AEIMRSFSLSTNLQESLRSKE
[0014] The protein has considerable clinical importance as a broad
spectrum anti-viral, anti-proliferative and immunomodulating agent.
Recombinant and other preparations of INF.alpha.2 have been used
therapeutically in a variety of cancer and viral indications in man
[reviewed in Sen, G. G. and Lengyel P, (1992), J. Biol. Chem. 267:
5017-5020]. However despite very significant therapeutic benefit to
large numbers of patients, resistance to therapy in certain
patients has been documented and one important mechanism of
resistance has been shown to be the development of neutralising
antibodies detectable in the serum of treated patients [Quesada, J.
R. et al (1985) J. Clin. Oncology 3:1522-1528; Stein R. G. et al
(1988) ibid; Russo, D. et al (1996) ibid; Brooks M. G. et al (1989)
Gut 30: 1116-1122]. An immune response in these patients is mounted
to the therapeutic interferon despite the fact that a molecule of
at least identical primary structure is produced endogenously in
man.
[0015] Others have provided modified INF.alpha.2a and methods of
use [U.S. Pat. No. 4,496,537; U.S. Pat. No. 5,972,331; U.S. Pat.
No. 5,480,640; U.S. Pat. No. 5,190,751; U.S. Pat. No. 4,959,210],
but these approaches have been directed towards improvements in the
commercial production of INF.alpha.2a. Such teachings do not
recognize the importance of T-cell epitopes to the immunogenic
properties of the protein nor have been conceived to directly
influence said properties in a specific and controlled way
according to the scheme of the present invention.
[0016] However, there is a continued need for INF.alpha.2a
analogues with enhanced properties. Desired enhancements include
alternative schemes and modalities for the expression and
purification of the said therapeutic, but also and especially,
improvements in the biological properties of the protein. There is
a particular need for enhancement of the in vivo characteristics
when administered to the human subject. In this regard, it is
highly desired to provide INF.alpha.2a with reduced or absent
potential to induce an immune response in the human subject.
SUMMARY AND DESCRIPTION OF THE INVENTION
[0017] The present invention provides for modified forms of human
interferon .alpha., and specifically the interferon .alpha.2 type,
herein called "INF.alpha.2", in which the immune characteristic is
modified by means of reduced or removed numbers of potential T-cell
epitopes.
[0018] The invention discloses sequences identified within the
INF.alpha.2 primary sequence that are potential T-cell epitopes by
virtue of MHC class II binding potential. This disclosure
specifically pertains the human INF.alpha.2 protein being 165 amino
acid residues. The invention discloses also specific positions
within the primary sequence of the molecule which according to the
invention are to be altered by specific amino acid substitution,
addition or deletion without in principal affecting the biological
activity. In cases in which the loss of immunogenicity can be
achieved only by a simultaneous loss of biological activity it is
possible to restore said activity by further alterations within the
amino acid sequence of the protein.
[0019] The invention furthermore discloses methods to produce such
modified molecules, and above all methods to identify said T-cell
epitopes which require alteration in order to reduce or remove
immunogenic sites.
[0020] The protein according to this invention would expect to
display an increased circulation time within the human subject and
would be of particular benefit in chronic or recurring disease
settings such as is the case for a number of indications for
INF.alpha.2. The present invention provides for modified forms of
INF.alpha.2 proteins that are expected to display enhanced
properties in vivo. These modified INF.alpha.2 molecules can be
used in pharmaceutical compositions.
[0021] In summary the invention relates to the following issues:
[0022] a modified molecule having the biological activity of human
interferon alpha 2 (INF.alpha.2) and being substantially
non-immunogenic or less immunogenic than any non-modified molecule
having the same biological activity when used in vivo; [0023] a
corresponding molecule, wherein said loss of immunogenicity is
achieved by removing one or more T-cell epitopes, preferably one
T-cell epitope, derived from the originally non-modified molecule
and/or by reduction in numbers of MHC allotypes able to bind
peptides derived from said molecule; [0024] a corresponding
molecule, wherein said originally present T-cell epitopes are MHC
class II ligands or peptide sequences which show the ability to
stimulate or bind T-cells via presentation on MHC class II; [0025]
a corresponding molecule, wherein said ligands or peptide sequences
are 13 mer or 15 mer peptides; [0026] a correspondingly molecule,
wherein said peptide sequences are selected from the group as
depicted in FIG. 1. [0027] a corresponding molecule, wherein 1-9
amino acid residues, preferably one amino acid residue, in any of
the originally present T-cell epitopes are altered; [0028] a
corresponding molecule, wherein the alteration of the amino acid
residues is substitution, addition or deletion, preferably
substitution, of originally present amino acid(s) residue(s) by
other amino acid residue(s) at specific position(s); [0029] a
corresponding molecule, wherein one or more of the amino acid
residue substitutions are made as indicated in FIG. 2, and, in
addition, optionally one or more of the amino acid residue
substitutions are carried out as indicated in FIG. 3 for the
reduction in the number of MHC allotypes able to bind peptides
derived from said molecule; [0030] a corresponding molecule,
wherein additionally further alteration, such as substitution,
addition or deletion is conducted to restore biological activity of
said molecule; [0031] a corresponding modified molecule, wherein
the amino acid alteration is made with reference to an homologous
protein sequence or with reference to in silico modeling
techniques;
[0032] a modified molecule having the biological activity of human
interferon alpha 2 (INF.alpha.2) and being substantially
non-immunogenic or less immunogenic than any non-modified molecule
having the same biological activity when used in vivo, obtainable
by alteration of one or more amino acids in the primary sequence by
(i) removing one or more T-cell epitopes derived from the
originally non-modified molecule and being MHC class II ligands or
peptide sequences which show the ability to stimulate or bind
T-cells via presentation on MHC class II, and/or (ii) by reduction
in numbers of MHC allotypes able to bind peptides derived from said
molecule, wherein said modified molecule comprises alterations
which are made at one or more positions within-. following strings
of contiguous amino acid residues of said primary sequence derived
from the INF.quadrature.2 wild-type: TABLE-US-00002 (a)
ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLH (R1), (b) FNLFSTKDSSAAWDE (R2),
(c) KEDSILAVRKYFQRITLY (R3);
[0033] a corresponding molecule, wherein said alteration is
substitution of 1-9 amino acid residues; [0034] a corresponding
molecule, wherein said substitution is conducted at one or more
amino acid residues from the strings R1, R2 and R3, preferably R2
and R3, and more preferably R3; [0035] a corresponding molecule,
wherein additionally one or more substitutions of amino acid
residues outside the sequence strings R1, R2 or R3 are conducted;
[0036] a corresponding molecule comprising an amino acid residue
substitution made at one or more positions in the wild-type
molecule: 24, 26, 27, 38, 55, 63, 64, 66, 67, 76, 84, 85, 89, 103,
110, 111, 116, 117, 119, 122, 123, 126, 128, 129, 130, 153,
preferably at one or more of the following positions 26, 27, 38,
63, 85, 89, 103, 110, 111, 116, 117, 122, 123, 126, 128, 153, more
preferably 103, 110, 111, 116, 117, 122, 123, 126, 128, 153; [0037]
a preferred embodiment, wherein said substitution is made at one or
more positions selected from 26, 27, 38 and additionally at one or
more positions selected from 103, 110, 111, 116, 117, 122, 123,
126, 128, 153, or alternatively, selected from 63, 85, 89 and 103,
110, 111, 116, 117, 122, 123, 126, 128, 153; [0038] a corresponding
molecule, wherein said substitution is made at one or more
positions as specified in FIG. 4; [0039] a corresponding molecule,
wherein said substitution is made at positions L26P, F27S, F38E
and/or I63T, Y85S, Y89D, Y89E, Y89N and/or V103E, L110G, M111T,
M111S, M111E, I116S, I116Q, L117G, L117A, Y122E, Y122Q, F123H,
I126A, L128A, L153S; [0040] a preferred corresponding molecule
wherein said substitution is made at positions L26P, F27S, F38E
and/or I63T, Y85S, Y89D, Y89E, Y89N and/or V103E, L110G, M111T,
M111S, M111E, I116S, I116Q, L117G, L117A; [0041] a modified
molecule having the biological activity of human interferon alpha 2
(INF.alpha.2) and being substantially non-immunogenic or less
immunogenic than any non-modified molecule having the same
biological activity when used in vivo, obtainable by substitution
of one or more amino acids in the primary sequence by (i) removing
one or more T-cell epitopes derived from the originally
non-modified molecule and being MHC class II ligands or peptide
sequences which show the ability to stimulate or bind T-cells via
presentation on MHC class II, and/or (ii) by reduction in numbers
of MHC allotypes able to bind peptides derived from said molecule,
wherein said substitution is made at one or more positions in a
wild-type molecule INF.alpha.2a or INF.alpha.2b corresponding to at
least one of the groups selected from:
[0042] (i) I24P, L26P, F27S, F38E, V55A,
[0043] (ii) I63T, L66A, F67D, F67E, W76H, F84D, F84E, Y85S, Y89D,
Y89E, Y89N,
[0044] (iii) any position within sequence R3; [0045] a
corresponding molecule, whereby one or more of the following
substitutions are made within sequence R3: V103E, L110G, L110S,
M111T, M111S, M111E, I116S, I116Q, L117G, L117A, V119A, Y122Q,
Y122E, Y122H, F123H, I126A, L128A, Y129N, L130G, L130, L153S,
preferably Y122E, Y122Q, F123H, I126A, L128A, optionally containing
additional amino acid residue alterations, preferably
substitutions, which lead to a further diminished
immunogenicity;
[0046] a modified human interferon alpha 2 (INF.alpha.2) having
reduced immunogenicity consisting of the following sequence:
TABLE-US-00003
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDRHDFGFPQEEFGNQFQK
AETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACV
IQGVGVTETPLMKEDSILAVRKX.sup.1
X.sup.2QRX.sup.3TX.sup.4YLKEKKYSPCAWEVVRA EIMRSFSLSTNLQESLRSKE,
wherein [0047] X.sup.0 is R, K; [0048] X.sup.1 is Y, E, Q; [0049]
X.sup.2 is F, H; [0050] X.sup.3 is I, A; and [0051] X.sup.4is L, A;
whereby simultaneously X.sup.1=Y, X.sup.2=F, X.sup.3=I and
X.sup.4=L are excluded (this sequence corresponds to the wild-type
IFN.alpha.2);
[0052] a modified human interferon alpha 2 (INF.alpha.2) having
reduced immunogenicity consisting of the following sequence:
TABLE-US-00004
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDRHDFGFPQEEFGNQFQK
AETIPVLHEMIQQIFNJLFSTKDSSAAWDETLLDKFYTELYQQLNDLEAC
VIQGVGVTETPX.sup.1X.sup.2KEDSX.sup.3X.sup.4AVRKX.sup.5X.sup.6QRX.sup.7TX.s-
up.8YLKEKKYSPCAW EVVRAEIMRSFSX9STNLQESLRSKE,
wherein [0053] X.sup.0 is R, K; [0054] X.sup.1 is L, S, G, [0055]
X.sup.2 is M, T, S, E, [0056] X.sup.3 is I, S, Q, [0057] X.sup.4 is
L, G, [0058] X.sup.5 is Y, E, Q; [0059] X.sup.6 is F, H; [0060]
X.sup.7 is I, A; [0061] X.sup.8 is L, A; and, [0062] X.sup.9 is L,
S whereby simultaneously X.sup.1=L, X.sup.2=M, X.sup.3=I,
X.sup.4=L, X.sup.5=Y, X.sup.6=F, X.sup.7=I, X.sup.8=L and X.sup.9=L
are excluded;
[0063] a modified human interferon alpha 2 (INF.alpha.2) having
reduced immunogenicity consisting of the following sequence:
TABLE-US-00005
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDFGFPQEEFGNQFQKAET
IPVLHEMIQQX.sup.1X.sup.2NX.sup.3X.sup.4STKDSSAAX.sup.5DETLLDKX.sup.6X.sup.-
7TELX.sup.8QQLND LEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVR
AEIMRSFSLSTNLQESLRSKE,
wherein [0064] X.sup.0 is R, K; [0065] X.sup.1 is I, T; [0066]
X.sup.2 is F, D, A; [0067] X.sup.3 is L, A; [0068] X.sup.4 is F, D,
E; [0069] X.sup.5 is W, H; [0070] X.sup.6 is F, D, E; [0071]
X.sup.7 is Y, S and [0072] X.sup.8 is Y, D, E, N; whereby
simultaneously X.sup.1=I, X.sup.2=F, X.sup.3=L, X.sup.4=F,
X.sup.5=W, X.sup.6=F, X.sup.7=Y and X.sup.8=Y are excluded;
[0073] a modified human interferon alpha 2 (INF.alpha.2) having
reduced immunogenicity consisting of the following sequence:
TABLE-US-00006
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISLFSCLKDRHDFGFPQEEFGNQFQK
AETIPVLHEMIQQX.sup.1FNLFSTKDSSAAWDETLLDKFX.sup.2TELX.sup.3QQLNDLE
ACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAE
IMRSFSLSTNLQESLRSKE,
wherein [0074] X.sup.0 is R, K; [0075] X.sup.1 is I, T; [0076]
X.sup.2 is Y, S and [0077] X.sup.3 is Y, D, E, N; whereby
simultaneously X.sup.1=I, X.sup.2=Y and X.sup.3=Y are excluded;
[0078] a modified human interferon alpha 2 (INF.alpha.2) having
reduced immunogenicity consisting of the following sequence:
TABLE-US-00007
CDLPQTHSLGSRRTLMLLAQMRX.sup.0ISX.sup.1X.sup.2SCLKDRHDFGX.sup.3PQEEFGNQ
FQKAETIPX.sup.4LHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDL
EACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRA
EIMRSFSLSTNLQESLRSKE,
wherein [0079] X.sup.0 is R, K; [0080] X.sup.1 is; L, P, [0081]
X.sup.2 is; F, S, [0082] X.sup.3 is F, E and [0083] X.sup.4 is; V,
A whereby simultaneously X.sup.1=L, X.sup.2=F, X.sup.3=F and
X.sup.4=V are excluded; it should be pointed out that all exclusion
specified in the above formulas refer to and shall include all
non-deimmunized versions of IFN.alpha.2 which are known in the
prior art; [0084] corresponding modified INF.alpha.2 sequences,
wherein additional substitutions are made especially by the
combinations as indicated in the claims; [0085] a corresponding
modified INF.alpha.2 sequence, wherein additional substitutions are
made, preferably at one ore more positions within partial sequence
R1 and/or R2 and /or R3 (whereby R1, R2, R3 are defined as
indicated above); [0086] a DNA sequence coding for a modified
INF.alpha.2 as described above and below; [0087] a pharmaceutical
composition comprising a modified molecule having the biological
activity of INF.alpha.2 as defined above, optionally together with
a pharmaceutically acceptable carrier, diluent or excipient; [0088]
a method for manufacturing a modified molecule having the
biological activity of INF.alpha.2 as defined above and below
comprising the following steps: (i) determining the amino acid
sequence of the polypeptide or part thereof,
[0089] (ii) identifying one or more potential T-cell epitopes
within the amino acid sequence of the protein by any method
including determination of the binding of the peptides to MHC
molecules using in vitro or in silico techniques or biological
assays, (iii) designing new sequence variants with one or more
amino acids within the identified potential T-cell epitopes
modified in such a way to substantially reduce or eliminate the
activity of the T-cell epitope as determined by the binding of the
peptides to MHC molecules using in vitro or in silico techniques or
biological assays ,or by binding of peptide-MHC complexes to
T-cells, (iv) constructing such sequence variants by recombinant
DNA techniques and testing said variants in order to identify one
or more variants with desirable properties, and (v) optionally
repeating steps (ii)-(iv); [0090] a corresponding method, wherein
step (iii) is carried out by substitution, addition or deletion of
1-9 amino acid residues in any of the originally present T-cell
epitopes or with reference to an homologous protein sequence and/or
in silico modeling techniques; [0091] a corresponding method,
wherein step (ii) is carried out by the following steps: (a)
selecting a region of the peptide having a known amino acid residue
sequence; (b) sequentially sampling overlapping amino acid residue
segments of predetermined uniform size and constituted by at least
three amino acid residues from the selected region; (c) calculating
MHC Class II molecule binding score for each said sampled segment
by summing assigned values for each hydrophobic amino acid residue
side chain present in said sampled amino acid residue segment; and
(d) identifying at least one of said segments suitable for
modification, based on the calculated MHC Class II molecule binding
score for that segment, to change overall MHC Class II binding
score for the peptide without substantially the reducing
therapeutic utility of the peptide; [0092] a corresponding method,
wherein step (c) is carried out by using a Bohm scoring function
modified to include 12-6 van der Waal's ligand-protein energy
repulsive term and ligand conformational energy term by (1)
providing a first data base of MHC Class II molecule models; (2)
providing a second data base of allowed peptide backbones for said
MHC Class II molecule models; (3) selecting a model from said first
data base; (4) selecting an allowed peptide backbone from said
second data base; (5) identifying amino acid residue side chains
present in each sampled segment; (6) determining the binding
affinity value for all side chains present in each sampled segment;
and repeating steps (1) through (5) for each said model and each
said backbone; [0093] a peptide molecule, which is a T-cell
epitope, consisting of 13 consecutive amino acid residues having a
potential MHC class II binding activity and created from the
primary sequence of non-modified INF.alpha.2, selected from the
group as depicted in FIG. 1, FIG. 6a-c; [0094] a peptide molecule
consisting of 15, preferably at least 9, consecutive amino acid
residues having a potential MHC class II binding activity and
created from the primary sequence of non-modified INFa2, selected
from any of the groups of partial sequences R1, R2, R3 or selected
from FIG. 7; [0095] a peptide molecule consisting of 9 -15
consecutive amino acid residues, having a potential MHC class II
binding activity and created from the primary sequence of
non-modified INF.alpha.2, whereby said molecule has a stimulation
index of at least 1.8, preferably 1.8-2, more preferably>2, in a
biological assay of cellular proliferation, wherein said index is
taken as the value of cellular proliferation scored following
stimulation by a peptide and divided by the value of cellular
proliferation scored in control cells not in receipt peptide and
wherein cellular proliferation is measured by any suitable means
according to standard methods as described in more detail in the
Examples; [0096] a corresponding peptide molecule having such
stimulation index value consisting of any of the sequence as
indicated in FIG. 6 or 7; [0097] a corresponding peptide molecule
having such stimulation index value comprising at least 9
consecutive amino acid residues from any of the INF.alpha.2 partial
sequences R1, R2, R3 as defined in claim above; [0098] a use of a
corresponding peptide, for the manufacture of INF.alpha.2 having
substantially no or less immunogenicity than any non-modified
molecule with the same or acceptably reduced degree of biological
activity when used in vivo; [0099] a pharmaceutical composition
consisting of a synthetic peptide sequence as specified above and
below and in the Figures having the biological activity of
IFN.alpha.2, optionally together with a pharmaceutically acceptable
carrier, diluent or excipient.
[0100] The term "T-cell epitope" means according to the
understanding of this invention an amino acid sequence which is
able to bind MHC class II, able to stimulate T-cells and/or also to
bind (without necessarily measurably activating) T-cells in complex
with MHC class II.
[0101] The term "peptide" as used herein and in the appended
claims, is a compound that includes two or more amino acids. The
amino acids are linked together by a peptide bond (defined herein
below). There are 20 different naturally occurring amino acids
involved in the biological production of peptides, and any number
of them may be linked in any order to form a peptide chain or ring.
The naturally occurring amino acids employed in the biological
production of peptides all have the L-configuration. Synthetic
peptides can be prepared employing conventional synthetic methods,
utilizing L-amino acids, D-amino acids, or various combinations of
amino acids of the two different configurations. Some peptides
contain only a few amino acid units. Short peptides, e.g., having
less than ten amino acid units, are sometimes referred to as
"oligopeptides". Other peptides contain a large number of amino
acid residues, e.g. up to 100 or more, and are referred to as
"polypeptides". By convention, a "polypeptide" may be considered as
any peptide chain containing three or more amino acids, whereas a
"oligopeptide" is usually considered as a particular type of
"short" polypeptide. Thus, as used herein, it is understood that
any reference to a "polypeptide" also includes an oligopeptide.
Further, any reference to a "peptide" includes polypeptides,
oligopeptides, and proteins. Each different arrangement of amino
acids forms different polypeptides or proteins. The number of
polypeptides--and hence the number of different proteins--that can
be formed is practically unlimited. "Alpha carbon (C.alpha.)" is
the carbon atom of the carbon-hydrogen (CH) component that is in
the peptide chain. A "side chain" is a pendant group to C.alpha.
that can comprise a simple or complex group or moiety, having
physical dimensions that can vary significantly compared to the
dimensions of the peptide.
[0102] The invention may be applied to any INF.alpha.2 species of
molecule with substantially the same primary amino acid sequences
as those disclosed herein and would include therefore INF.alpha.2
molecules derived by genetic engineering means or other processes
and may contain more or less than 165 amino acid residues.
[0103] INF.alpha.2 proteins such as identified from other mammalian
sources have in common many of the peptide sequences of the present
disclosure and have in common many peptide sequences with
substantially the same sequence as those of the disclosed listing.
Such protein sequences equally therefore fall under the scope of
the present invention. The invention is conceived to overcome the
practical reality that soluble proteins introduced into autologous
organisms can trigger an immune response resulting in development
of host antibodies that bind to the soluble protein. A prominent
example of this phenomenon amongst others, is the clinical use
INF.alpha.2. A significant proportion of human patients treated
with INF.alpha.2 make antibodies despite the fact that this protein
is produced endogenously [Russo, D. et al (1996) ibid; Stein, R. et
al (1988) ibid]. The present invention seeks to address this by
providing INF.alpha.2 proteins with altered propensity to elicit an
immune response on administration to the human host. According to
the methods described herein, the inventors have discovered and now
disclose the regions of the INFa2 molecule comprising the critical
T-cell epitopes driving the immune responses to this autologous
protein.
[0104] The general method of the present invention leading to the
modified INF.alpha.2 comprises the following steps:
[0105] (a) determining the amino acid sequence of the polypeptide
or part thereof;
[0106] (b) identifying one or more potential T-cell epitopes within
the amino acid sequence of the protein by any method including
determination of the binding of the peptides to MHC molecules using
in vitro or in silico techniques or biological assays;
[0107] (c) designing new sequence variants with one or more amino
acids within the identified potential T-cell epitopes modified in
such a way to substantially reduce or eliminate the activity of the
T-cell epitope as determined by the binding of the peptides to MHC
molecules using in vitro or in silico techniques or biological
assays. Such sequence variants are created in such a way to avoid
creation of new potential T-cell epitopes by the sequence
variations unless such new potential T-cell epitopes are, in turn,
modified in such a way to substantially reduce or eliminate the
activity of the T-cell epitope; and
[0108] (d) constructing such sequence variants by recombinant DNA
techniques and testing said variants in order to identify one or
more variants with desirable properties according to well known
recombinant techniques.
[0109] The identification of potential T-cell epitopes according to
step (b) can be carried out according to methods describes
previously in the prior art. Suitable methods are disclosed in WO
98/59244; WO 98/52976; WO 00/34317 and may preferably be used to
identify binding propensity of INF.alpha.2a -derived peptides to an
MHC class II molecule.
[0110] Another very efficacious method for identifying T-cell
epitopes by calculation is described in the EXAMPLE 1 which is a
preferred embodiment according to this invention.
[0111] In practice a number of variant INF.alpha.2 proteins will be
produced and tested for the desired immune and functional
characteristic. The variant proteins will most preferably be
produced by recombinant DNA techniques although other procedures
including chemical synthesis of INF.alpha.2 fragments may be
contemplated.
[0112] The results of an analysis according to step (b) of the
above scheme and pertaining to the human INF.alpha.2a protein
sequence of 165 amino acid residues is presented in FIG. 1. The
results of a design and constructs according to step (c) and (d) of
the above scheme and pertaining to the modified molecule of this
invention is presented in FIGS. 2 and 3.
[0113] The invention relates to INF.alpha.2 analogues in which
substitutions of at least one amino acid residue have been made at
positions resulting in a substantial reduction in activity of or
elimination of one or more potential T-cell epitopes from the
protein. One or more amino acid substitutions at particular points
within any of the potential MHC class II ligands identified in
Table 1 may result in a INF.alpha.2 molecule with a reduced
immunogenic potential when administered as a therapeutic to the
human host.
[0114] It is most preferred to provide an INF.alpha.2 molecule in
which amino acid modification (e.g. a substitution) is conducted
within the most immunogenic regions of the parent molecule. The
inventors herein have discovered that the most immunogenic regions
of the INF.alpha.2 molecule in man are confined to three regions
R1, R2 and R3 comprising respectively amino acid sequences;
ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLH; FNLFSTKDSSAAWDE and
KEDSILAVRKYFQRITLY. The major preferred embodiments of the present
invention comprise INFa2 molecules for which the MHC class II
ligands of FIG. 1 and which align either in their entirety or to a
minimum of 9 amino acid residues with any of the above sequence
elements R1, R2 or R3 are altered such as to eliminate binding or
otherwise reduce the numbers of MHC allotypes to which the peptide
can bind.
[0115] The preferred embodiments of the invention include the
specific substitutions of FIG. 4. It is particularly preferred to
provide modified INF.alpha.2 molecules containing combinations of
substitutions from FIG. 4. Combinations which comprise modification
to each of the immunogenic regions R1, R2 and R3 are preferred, and
combinations comprising modifications to R2 and R3 are especially
preferred although such preference is not intended to limit the
combinations of substitution which are considered desirable.
[0116] For the elimination of T-cell epitopes, amino acid
substitutions are preferably made at appropriate points within the
peptide sequence predicted to achieve substantial reduction or
elimination of the activity of the T-cell epitope. In practice an
appropriate point will preferably equate to an amino acid residue
binding within one of the pockets provided within the MHC class II
binding groove.
[0117] It is most preferred to alter binding within the first
pocket of the cleft at the so-called P1 or P1 anchor position of
the peptide. The quality of binding interaction between the P1
anchor residue of the peptide and the first pocket of the MHC class
II binding groove is recognized as being a major determinant of
overall binding affinity for the whole peptide. An appropriate
substitution at this position of the peptide will be for a residue
less readily accommodated within the pocket, for example,
substitution to a more hydrophilic residue. Amino acid residues in
the peptide at positions equating to binding within other pocket
regions within the MHC binding cleft are also considered and fall
under the scope of the present.
[0118] It is understood that single amino acid substitutions within
a given potential T-cell epitope are the most preferred route by
which the epitope may be eliminated. Combinations of substitution
within a single epitope may be contemplated and for example can be
particularly appropriate where individually defined epitopes are in
overlap with each other. Moreover, amino acid substitutions either
singly within a given epitope or in combination within a single
epitope may be made at positions not equating to the "pocket
residues" with respect to the MHC class II binding groove, but at
any point within the peptide sequence. Substitutions may be made
with reference to an homologues structure or structural method
produced using in silico techniques known in the art and may be
based on known structural features of the molecule according to
this invention. All such substitutions fall within the scope of the
present invention.
[0119] Amino acid substitutions other than within the peptides
identified above may be contemplated particularly when made in
combination with substitution(s) made within a listed peptide. For
example a change may be contemplated to restore structure or
biological activity of the variant molecule. Such compensatory
changes and changes to include deletion or addition of particular
amino acid residues from the INF.alpha.2 polypeptide resulting in a
variant with desired activity and in combination with changes in
any of the disclosed peptides fall under the scope of the
present.
[0120] In as far as this invention relates to modified INF.alpha.2,
compositions containing such modified INF.alpha.2 proteins or
fragments of modified INF.alpha.2 proteins and related compositions
should be considered within the scope of the invention. In another
aspect, the present invention relates to nucleic acids encoding
modified INF.alpha.2 entities. In a further aspect the present
invention relates to methods for therapeutic treatment of humans
using the modified INF.alpha.2 proteins.
SHORT DESCRIPTION OF THE FIGURES
[0121] The invention will now be illustrated, but not limited, by
the following examples. The examples refer to the following
drawings. The amino acid residues are consequently depicted as
one-letter code.
[0122] FIG. 1 provides peptide sequences in human INF.alpha.2a with
potential human MHC class II binding activity.
[0123] FIG. 2 provides substitutions leading to the elimination of
T-cell epitopes of human INF.alpha.2a (WT=wild-type residue).
[0124] FIG. 3 provides additional substitutions leading to the
removal of a potential T-cell epitope for one or more MHC
allotypes.
[0125] FIG. 4 provides preferred substitutions in human
INF.alpha.2a (WT=wild-type residue, MUT=desired residue).
[0126] FIG. 5 provides a table of the INF.alpha.2 13-mer synthetic
peptides sequences analysed using an MHC class II in vitro binding
assay of EXAMPLE 2.
[0127] FIG. 6 shows the results of in vitro MHC peptide binding
assays for MHC allotypes. a) indicates peptides with high affinity
binding (0% inhibition by competitor reference peptide) for each of
the MHC allotypes tested; b) indicates peptides with medium
affinity (0-50% inhibition by competitor) binding for each of the
MHC allotypes tested; c) indicates peptides with low (50-100%
inhibition by competitor) affinity binding for each of the MHC
allotypes tested and d) indicates peptides with no detectable
binding to the MHC allotypes tested.
[0128] FIG. 7 provides a table of the INF.alpha.2 15-mer peptide
sequences analysed using the naive human in vitro T-cell assay of
EXAMPLE 3. The peptide ID# and position of the N-terminal peptide
residue within the INF.alpha.2 sequence is indictated.
[0129] FIG. 8 shows cumulative stimulation indexes from 6
individuals that respond to stimulation with IFN.alpha. peptides.
Six donors from 20 screened responded to stimulation with one or
more of 51 15 mer peptides from the IFN.alpha. sequence. Responses
to individual peptides are grouped into three distinct regions with
region three containing the most immunogenic peptides #38 and #39
(arrows). Control peptides C32 (DRB1-restricted) and C49
(DP-restricted) are included for comparison. Cross-hatching within
each bar indicates the contribution from individual donors.
[0130] FIG. 9 shows the immunogenic regions within INF.alpha. and
details the peptide sequences from these regions able to stimulate
naive human T-cells.
[0131] FIG. 10 provides a table indicating INF.alpha. peptides
capable of promoting proliferation of naive human T-cells in vitro.
For 5 of the donors, responses are recorded to multiple overlapping
peptides from the major epitope regions R1, R2 and R3. For 3 of the
donors, responses are recorded to individual synthetic peptides
from R1, R2 or R3.
[0132] FIG. 11 provides a table showing frequency of MHC class II
alleles in the responding and non-responding donors to IFN.alpha.
peptides. a=Numerator is number of donors with DR allele,
denominator is number of donors that showed T cell proliferation in
vitro to that peptide (total number of responding donors=6).
b=Frequency of allele in donor population. Peptides for which two
or fewer responses were recorded were not evaluated. All responding
donors tested negative for DRB1*14. The DRB1*14 allotype has a
frequency of 1.5% in the 20 donors tested. Allorestriction of a
given peptide is determined by the frequency of an allele in the
donor population and the number of responding donors that express
the same allele. If a peptide is associated with any particular
allele (allorestricted) then the percentage shown would be expected
to be greater than the frequency for the allele in the
population.
[0133] FIG. 12 provides tables of IC.sub.50 values for 15-mer
synthetic peptides in competition binding assay for particular MHC
class II allotypes.
[0134] (a) Competition MHC class II peptide binding assay to
determine relative binding affinities of INF.alpha. peptides
capable of promoting proliferation of naive human T-cells in vitro
to DRB1*0101. INF.alpha. peptides were incubated with fixed HOM-2
cells in the presence of 10 .mu.M biotinylated influenza
haemagglutinin 307-319. The concentration of competitor peptide
causing 50% inhibition of maximun biotinylated peptide binding was
taken as the IC.sub.50. Influenza 103-115 was included as a high
affinity control. IC.sub.50.ltoreq.20 .mu.M=high affinity,
IC.sub.50=20-100 .mu.M=Medium Affinity, IC.sub.50.gtoreq.100 uM=Low
Affinity.
[0135] (b) Competition MHC class II peptide binding assay to
determine relative binding affinities of INF.alpha. peptides
capable of promoting proliferation of naive human T-cells in vitro
to DRB1*0701. INF.alpha. peptides were incubated with fixed MOU
(MANN) cells in the presence of 10 uM biotinylated tetanus toxin
828-840. The concentration of competitor peptide causing 50%
inhibition of maximun biotinylated peptide binding was taken as the
IC.sub.50. Tetanus toxin 828-840 was included as a high affinity
control. IC.sub.50.ltoreq.20 .mu.M=high affinity, IC.sub.50=20-100
.mu.M=Medium Affinity, IC.sub.50.gtoreq.100 .mu.M=Low Affinity.
[0136] (c) Competition MHC class II peptide binding assay to
determine relative binding affinities of INF.alpha. peptides
capable of promoting proliferation of naive human T-cells in vitro
to DRB1*0401. INF.alpha. peptides were incubated with fixed WT-51
cells in the presence of 50 uM biotinylated influenza
haemagglutinin 307-319. The concentration of competitor peptide
causing 50% inhibition of maximun biotinylated peptide binding was
taken as the IC.sub.50. Influenza 103-115 was included as a high
affinity control. IC.sub.50.ltoreq.20 .mu.M=high affinity,
IC.sub.50=20-100 .mu.M=Medium Affinity, IC.sub.50.gtoreq.100
.mu.M=Low Affinity.
[0137] FIG. 13 provides a table detailing substitutions within
INF.alpha. which provide molecules with retained activity in the
anti-proliferation assay of EXAMPLE 7. WT=wild-type residue;
#=residue number; Mut=mutation conducted. Epitope Region indicates
location of substitution with respect to immunogenic epitope
regions R1, R2 or R3.
[0138] FIG. 14 provides representative data of the
anti-proliferative effect of selected mutant INF.alpha.2 molecules.
Assays were conducted according to the methods of EXAMPLE 7. Panel
a) shows activity of molecules with substitution within immunogenic
epitope R1. Panel b) shows activity of molecules with substitution
within immunogenic epitope R2. Panel c) shows activity of molecules
with substitution within immunogenic epitope R3.
[0139] FIG. 15 provides panels that show individual donor responses
to INF.alpha. synthetic peptides. Data from control peptides C32
(DRB1-restricted) and C49 (DP-restricted) are included for
comparison. Immunogenic regions R1, R2, R3 are indicated on
relevant panels. Threshold for positive stimulation index=2.
[0140] In the following Examples the invention is described in more
detail which shall not be interpreted as a limitation or
restriction.
EXAMPLE 1
[0141] There are a number of factors that play important roles in
determining the total structure of a protein or polypeptide. First,
the peptide bond, i.e., that bond which joins the amino acids in
the chain together, is a covalent bond. This bond is planar in
structure, essentially a substituted amide. An "amide" is any of a
group of organic compounds containing the grouping --CONH--.
[0142] The planar peptide bond linking C.alpha. of adjacent amino
acids may be represented as depicted below: ##STR1##
[0143] Because the O.dbd.C and the C--N atoms lie in a relatively
rigid plane, free rotation does not occur about these axes. Hence,
a plane schematically depicted by the interrupted line is sometimes
referred to as an "amide" or "peptide plane" plane wherein lie the
oxygen (O), carbon (C), nitrogen (N), and hydrogen (H) atoms of the
peptide backbone. At opposite corners of this amide plane are
located the C.alpha. atoms. Since there is substantially no
rotation about the O.dbd.C and C--N atoms in the peptide or amide
plane, a polypeptide chain thus comprises a series of planar
peptide linkages joining the C.alpha. atoms.
[0144] A second factor that plays an important role in defining the
total structure or conformation of a polypeptide or protein is the
angle of rotation of each amide plane about the common C.alpha.
linkage. The terms "angle of rotation" and "torsion angle" are
hereinafter regarded as equivalent terms. Assuming that the O, C,
N, and H atoms remain in the amide plane (which is usually a valid
assumption, although there may be some slight deviations from
planarity of these atoms for some conformations), these angles of
rotation define the N and R polypeptide's backbone conformation,
i.e., the structure as it exists between adjacent residues. These
two angles are known as .phi. and .psi.. A set of the angles
.phi..sub.1, .psi..sub.1, where the subscript represents a
particular residue of a polypeptide chain, thus effectively defines
the polypeptide secondary structure. The conventions used in
defining the .phi., .psi. angles, i.e., the reference points at
which the amide planes form a zero degree angle, and the definition
of which angle is .phi., and which angle is .psi., for a given
polypeptide, are defined in the literature (see, e.g., Ramachandran
et al. Adv. Prot. Chem. 23:283-437 (1968), at pages 285-94, which
pages are incorporated herein by reference).
[0145] The present method can be applied to any protein, and is
based in part upon the discovery that in humans the primary Pocket
1 anchor position of MHC Class II molecule binding grooves has a
well designed specificity for particular amino acid side chains.
The specificity of this pocket is determined by the identity of the
amino acid at position 86 of the beta chain of the MHC Class II
molecule. This site is located at the bottom of Pocket 1 and
determines the size of the side chain that can be accommodated by
this pocket. Marshall, K. W., J. Immunol., 152:4946-4956 (1994). If
this residue is a glycine, then all hydrophobic aliphatic and
aromatic amino acids (hydrophobic aliphatics being: valine,
leucine, isoleucine, methionine and aromatics being: phenylalanine,
tyrosine and tryptophan) can be accommodated in the pocket, a
preference being for the aromatic side chains. If this pocket
residue is a valine, then the side chain of this amino acid
protrudes into the pocket and restricts the size of peptide side
chains that can be accommodated such that only hydrophobic
aliphatic side chains can be accommodated. Therefore, in an amino
acid residue sequence, wherever an amino acid with a hydrophobic
aliphatic or aromatic side chain is found, there is the potential
for a MHC Class II restricted T-cell epitope to be present. If the
side-chain is hydrophobic aliphatic, however, it is approximately
twice as likely to be associated with a T-cell epitope than an
aromatic side chain (assuming an approximately even distribution of
Pocket 1 types throughout the global population).
[0146] A computational method embodying the present invention
profiles the likelihood of peptide regions to contain T-cell
epitopes as follows:
[0147] (1) The primary sequence of a peptide segment of
predetermined length is scanned, and all hydrophobic aliphatic and
aromatic side chains present are identified. (2) The hydrophobic
aliphatic side chains are assigned a value greater than that for
the aromatic side chains; preferably about twice the value assigned
to the aromatic side chains, e.g., a value of 2 for a hydrophobic
aliphatic side chain and a value of 1 for an aromatic side chain.
(3) The values determined to be present are summed for each
overlapping amino acid residue segment (window) of predetermined
uniform length within the peptide, and the total value for a
particular segment (window) is assigned to a single amino acid
residue at an intermediate position of the segment (window),
preferably to a residue at about the midpoint of the sampled
segment (window). This procedure is repeated for each sampled
overlapping amino acid residue segment (window). Thus, each amino
acid residue of the peptide is assigned a value that relates to the
likelihood of a T-cell epitope being present in that particular
segment (window). (4) The values calculated and assigned as
described in Step 3, above, can be plotted against the amino acid
coordinates of the entire amino acid residue sequence being
assessed. (5) All portions of the sequence which have a score of a
predetermined value, e.g., a value of 1, are deemed likely to
contain a T-cell epitope and can be modified, if desired.
[0148] This particular aspect of the present invention provides a
general method by which the regions of peptides likely to contain
T-cell epitopes can be described. Modifications to the peptide in
these regions have the potential to modify the MHC Class II binding
characteristics.
[0149] According to another aspect of the present invention, T-cell
epitopes can be predicted with greater accuracy by the use of a
more sophisticated computational method which takes into account
the interactions of peptides with models of MHC Class II alleles.
The computational prediction of T-cell epitopes present within a
peptide according to this particular aspect contemplates the
construction of models of at least 42 MHC Class II alleles based
upon the structures of all known MHC Class II molecules and a
method for the use of these models in the computational
identification of T-cell epitopes, the construction of libraries of
peptide backbones for each model in order to allow for the known
variability in relative peptide backbone alpha carbon (C.alpha.)
positions, the construction of libraries of amino-acid side chain
conformations for each backbone dock with each model for each of
the 20 amino-acid alternatives at positions critical for the
interaction between peptide and MHC Class II molecule, and the use
of these libraries of backbones and side-chain conformations in
conjunction with a scoring function to select the optimum backbone
and side-chain conformation for a particular peptide docked with a
particular MHC Class II molecule and the derivation of a binding
score from this interaction.
[0150] Models of MHC Class II molecules can be derived via homology
modeling from a number of similar structures found in the
Brookhaven Protein Data Bank ("PDB"). These may be made by the use
of semi-automatic homology modeling software (Modeller, Sali A.
& Blundell T L., 1993. J. Mol Biol 234:779-815) which
incorporates a simulated annealing function, in conjunction with
the CHARMm force-field for energy minimisation (available from
Molecular Simulations Inc., San Diego, Calif.). Alternative
modeling methods can be utilized as well.
[0151] The present method differs significantly from other
computational methods which use libraries of experimentally derived
binding data of each amino-acid alternative at each position in the
binding groove for a small set of MHC Class II molecules (Marshall,
K. W., et al., Biomed. Pept. Proteins Nucleic Acids, 1(3):157-162)
(1995) or yet other computational methods which use similar
experimental binding data in order to define the binding
characteristics of particular types of binding pockets within the
groove, again using a relatively small subset of MHC Class II
molecules, and then `mixing and matching` pocket types from this
pocket library to artificially create further `virtual` MHC Class
II molecules (Sturniolo T., et al., Nat. Biotech, 17(6): 555-561
(1999). Both prior methods suffer the major disadvantage that, due
to the complexity of the assays and the need to synthesize large
numbers of peptide variants, only a small number of MHC Class II
molecules can be experimentally scanned. Therefore the first prior
method can only make predictions for a small number of MHC Class II
molecules. The second prior method also makes the assumption that a
pocket lined with similar amino-acids in one molecule will have the
same binding characteristics when in the context of a different
Class II allele and suffers further disadvantages in that only
those MHC Class II molecules can be `virtually` created which
contain pockets contained within the pocket library. Using the
modeling approach described herein, the structure of any number and
type of MHC Class II molecules can be deduced, therefore alleles
can be specifically selected to be representative of the global
population. In addition, the number of MHC Class II molecules
scanned can be increased by making further models further than
having to generate additional data via complex experimentation.
[0152] The use of a backbone library allows for variation in the
positions of the C.alpha. atoms of the various peptides being
scanned when docked with particular MHC Class II molecules. This is
again in contrast to the alternative prior computational methods
described above which rely on the use of simplified peptide
backbones for scanning amino-acid binding in particular pockets.
These simplified backbones are not likely to be representative of
backbone conformations found in `real` peptides leading to
inaccuracies in prediction of peptide binding. The present backbone
library is created by superposing the backbones of all peptides
bound to MHC Class II molecules found within the Protein Data Bank
and noting the root mean square (RMS) deviation between the
C.alpha. atoms of each of the eleven amino-acids located within the
binding groove. While this library can be derived from a small
number of suitable available mouse and human structures (currently
13), in order to allow for the possibility of even greater
variability, the RMS figure for each C''-.alpha. position is
increased by 50%. The average C.alpha. position of each amino-acid
is then determined and a sphere drawn around this point whose
radius equals the RMS deviation at that position plus 50%. This
sphere represents all allowed C.alpha. positions.
[0153] Working from the C.alpha. with the least RMS deviation (that
of the amino-acid in Pocket 1 as mentioned above, equivalent to
Position 2 of the 11 residues in the binding groove), the sphere is
three-dimensionally gridded, and each vertex within the grid is
then used as a possible location for a C.alpha. of that amino-acid.
The subsequent amide plane, corresponding to the peptide bond to
the subsequent amino-acid is grafted onto each of these C.alpha.s
and the .phi. and .psi. angles are rotated step-wise at set
intervals in order to position the subsequent C.alpha.. If the
subsequent C.alpha. falls within the `sphere of allowed positions`
for this C.alpha. than the orientation of the dipeptide is
accepted, whereas if it falls outside the sphere then the dipeptide
is rejected.
[0154] This process is then repeated for each of the subsequent
C.alpha. positions, such that the peptide grows from the Pocket 1
C.alpha. `seed`, until all nine subsequent C.alpha.s have been
positioned from all possible permutations of the preceding
C.alpha.s. The process is then repeated once more for the single
C.alpha. preceding pocket 1 to create a library of backbone
C.alpha. positions located within the binding groove.
[0155] The number of backbones generated is dependent upon several
factors: The size of the `spheres of allowed positions`; the
fineness of the gridding of the `primary sphere` at the Pocket 1
position; the fineness of the step-wise rotation of the .phi. and
.psi. angles used to position subsequent C.alpha.s. Using this
process, a large library of backbones can be created. The larger
the backbone library, the more likely it will be that the optimum
fit will be found for a particular peptide within the binding
groove of an MHC Class II molecule. Inasmuch as all backbones will
not be suitable for docking with all the models of MHC Class II
molecules due to clashes with amino-acids of the binding domains,
for each allele a subset of the library is created comprising
backbones which can be accommodated by that allele.
[0156] The use of the backbone library, in conjunction with the
models of MHC Class II molecules creates an exhaustive database
consisting of allowed side chain conformations for each amino-acid
in each position of the binding groove for each MHC Class II
molecule docked with each allowed backbone. This data set is
generated using a simple steric overlap function where a MHC Class
II molecule is docked with a backbone and an amino-acid side chain
is grafted onto the backbone at the desired position. Each of the
rotatable bonds of the side chain is rotated step-wise at set
intervals and the resultant positions of the atoms dependent upon
that bond noted. The interaction of the atom with atoms of
side-chains of the binding groove is noted and positions are either
accepted or rejected according to the following criteria: The sum
total of the overlap of all atoms so far positioned must not exceed
a pre-determined value. Thus the stringency of the conformational
search is a function of the interval used in the step-wise rotation
of the bond and the pre-determined limit for the total overlap.
This latter value can be small if it is known that a particular
pocket is rigid, however the stringency can be relaxed if the
positions of pocket side-chains are known to be relatively
flexible. Thus allowances can be made to imitate variations in
flexibility within pockets of the binding groove. This
conformational search is then repeated for every amino-acid at
every position of each backbone when docked with each of the MHC
Class II molecules to create the exhaustive database of side-chain
conformations.
[0157] A suitable mathematical expression is used to estimate the
energy of binding between models of MHC Class II molecules in
conjunction with peptide ligand conformations which have to be
empirically derived by scanning the large database of
backbone/side-chain conformations described above. Thus a protein
is scanned for potential T-cell epitopes by subjecting each
possible peptide of length varying between 9 and 20 amino-acids
(although the length is kept constant for each scan) to the
following computations: An MHC Class II molecule is selected
together with a peptide backbone allowed for that molecule and the
side-chains corresponding to the desired peptide sequence are
grafted on. Atom identity and interatomic distance data relating to
a particular side-chain at a particular position on the backbone
are collected for each allowed conformation of that amino-acid
(obtained from the database described above). This is repeated for
each side-chain along the backbone and peptide scores derived using
a scoring function. The best score for that backbone is retained
and the process repeated for each allowed backbone for the selected
model. The scores from all allowed backbones are compared and the
highest score is deemed to be the peptide score for the desired
peptide in that MHC Class II model. This process is then repeated
for each model with every possible peptide derived from the protein
being scanned, and the scores for peptides versus models are
displayed.
[0158] In the context of the present invention, each ligand
presented for the binding affinity calculation is an amino-acid
segment selected from a peptide or protein as discussed above.
Thus, the ligand is a selected stretch of amino acids about 9 to 20
amino acids in length derived from a peptide, polypeptide or
protein of known sequence. The terms "amino acids" and "residues"
are hereinafter regarded as equivalent terms.
[0159] The ligand, in the form of the consecutive amino acids of
the peptide to be examined grafted onto a backbone from the
backbone library, is positioned in the binding cleft of an MHC
Class II molecule from the MHC Class II molecule model library via
the coordinates of the C''-.alpha. atoms of the peptide backbone
and an allowed conformation for each side-chain is selected from
the database of allowed conformations. The relevant atom identities
and interatomic distances are also retrieved from this database and
used to calculate the peptide binding score. Ligands with a high
binding affinity for the MHC Class II binding pocket are flagged as
candidates for site-directed mutagenesis. Amino-acid substitutions
are made in the flagged ligand (and hence in the protein of
interest) which is then retested using the scoring function in
order to determine changes which reduce the binding affinity below
a predetermined threshold value. These changes can then be
incorporated into the protein of interest to remove T-cell
epitopes.
[0160] Binding between the peptide ligand and the binding groove of
MHC Class II molecules involves non-covalent interactions
including, but not limited to: hydrogen bonds, electrostatic
interactions, hydrophobic (lipophilic) interactions and Van der
Walls interactions. These are included in the peptide scoring
function as described in detail below.
[0161] It should be understood that a hydrogen bond is a
non-covalent bond which can be formed between polar or charged
groups and consists of a hydrogen atom shared by two other atoms.
The hydrogen of the hydrogen donor has a positive charge where the
hydrogen acceptor has a partial negative charge. For the purposes
of peptide/protein interactions, hydrogen bond donors may be either
nitrogens with hydrogen attached or hydrogens attached to oxygen or
nitrogen. Hydrogen bond acceptor atoms may be oxygens not attached
to hydrogen, nitrogens with no hydrogens attached and one or two
connections, or sulphurs with only one connection. Certain atoms,
such as oxygens attached to hydrogens or imine nitrogens (e.g.
C.dbd.NH) may be both hydrogen acceptors or donors. Hydrogen bond
energies range from 3 to 7 Kcal/mol and are much stronger than Van
der Waal's bonds, but weaker than covalent bonds. Hydrogen bonds
are also highly directional and are at their strongest when the
donor atom, hydrogen atom and acceptor atom are co-linear.
[0162] Electrostatic bonds are formed between oppositely charged
ion pairs and the strength of the interaction is inversely
proportional to the square of the distance between the atoms
according to Coulomb's law. The optimal distance between ion pairs
is about 2.8 .ANG.. In protein/peptide interactions, electrostatic
bonds may be formed between arginine, histidine or lysine and
aspartate or glutamate. The strength of the bond will depend upon
the pKa of the ionizing group and the dielectric constant of the
medium although they are approximately similar in strength to
hydrogen bonds.
[0163] Lipophilic interactions are favorable
hydrophobic-hydrophobic contacts that occur between he protein and
peptide ligand. Usually, these will occur between hydrophobic amino
acid side chains of the peptide buried within the pockets of the
binding groove such that they are not exposed to solvent. Exposure
of the hydrophobic residues to solvent is highly unfavorable since
the surrounding solvent molecules are forced to hydrogen bond with
each other forming cage-like clathrate structures. The resultant
decrease in entropy is highly unfavorable. Lipophilic atoms may be
sulphurs which are neither polar nor hydrogen acceptors and carbon
atoms which are not polar.
[0164] Van der Waal's bonds are non-specific forces found between
atoms which are 3-4 .ANG. apart. They are weaker and less specific
than hydrogen and electrostatic bonds. The distribution of
electronic charge around an atom changes with time and, at any
instant, the charge distribution is not symmetric. This transient
asymmetry in electronic charge induces a similar asymmetry in
neighboring atoms. The resultant attractive forces between atoms
reaches a maximum at the Van der Waal's contact distance but
diminishes very rapidly at about 1 .ANG. to about 2 .ANG..
Conversely, as atoms become separated by less than the contact
distance, increasingly strong repulsive forces become dominant as
the outer electron clouds of the atoms overlap. Although the
attractive forces are relatively weak compared to electrostatic and
hydrogen bonds (about 0.6 Kcal/mol), the repulsive forces in
particular may be very important in determining whether a peptide
ligand may bind successfully to a protein.
[0165] In one embodiment, the Bohm scoring function (SCORE1
approach) is used to estimate the binding constant. (Bohm, H. J.,
J. Comput Aided Mol. Des., 8(3):243-256 (1994) which is hereby
incorporated in its entirety). In another embodiment, the scoring
function (SCORE2 approach) is used to estimate the binding
affinities as an indicator of a ligand containing a T-cell epitope
(Bohm, H. J., J. Comput Aided Mol. Des., 12(4):309-323 (1998) which
is hereby incorporated in its entirety). However, the Bohm scoring
functions as described in the above references are used to estimate
the binding affinity of a ligand to a protein where it is already
known that the ligand successfully binds to the protein and the
protein/ligand complex has had its structure solved, the solved
structure being present in the Protein Data Bank ("PDB").
Therefore, the scoring function has been developed with the benefit
of known positive binding data. In order to allow for
discrimination between positive and negative binders, a repulsion
term must be added to the equation. In addition, a more
satisfactory estimate of binding energy is achieved by computing
the lipophilic interactions in a pairwise manner rather than using
the area based energy term of the above Bohm functions.
[0166] Therefore, in a preferred embodiment, the binding energy is
estimated using a modified Bohm scoring function. In the modified
Bohm scoring function, the binding energy between protein and
ligand (.DELTA.G.sub.bind) is estimated considering the following
parameters: The reduction of binding energy due to the overall loss
of translational and rotational entropy of the ligand
(.DELTA.G.sub.0); contributions from ideal hydrogen bonds
(.DELTA.G.sub.hb) where at least one partner is neutral;
contributions from unperturbed ionic interactions
(.DELTA.G.sub.ionic); lipophilic interactions between lipophilic
ligand atoms and lipophilic acceptor atoms (.DELTA.G.sub.lipo); the
loss of binding energy due to the freezing of internal degrees of
freedom in the ligand, i.e., the freedom of rotation about each
C--C bond is reduced (.DELTA.G.sub.rot); the energy of the
interaction between the protein and ligand (E.sub.VdW).
Consideration of these terms gives equation 1:
(.DELTA.G.sub.bind)=(.DELTA.G.sub.0)+(.DELTA.G.sub.hb.times.N.sub.hb)+(.D-
ELTA.G.sub.ionic.times.N.sub.ionic)+(.DELTA.G.sub.lipo.times.N.sub.lipo)+(-
.DELTA.G.sub.rot+N.sub.rot)+(E.sub.VdW).
[0167] Where N is the number of qualifying interactions for a
specific term and, in one embodiment, .DELTA.G.sub.0,
.DELTA.G.sub.hb, .DELTA.G.sub.ionic, .DELTA.G.sub.lipo and
.DELTA.G.sub.rot are constants which are given the values: 5.4,
-4.7, -4.7, -0.17, and 1.4, respectively.
[0168] The term N.sub.hb is calculated according to equation 2:
N.sub.hb.SIGMA..sub.h-bondsf(.DELTA.R,
.DELTA..alpha.).times.f(N.sub.neighb).times.f.sub.pcs
[0169] f(.DELTA.R, .DELTA..alpha.) is a penalty function which
accounts for large deviations of hydrogen bonds from ideality and
is calculated according to equation 3: f .function. ( .DELTA.
.times. .times. R , .DELTA. - .alpha. ) = f1 .function. ( .DELTA.
.times. .times. R ) .times. f2 .function. ( .DELTA..alpha. )
##EQU1## Where .times. : .times. f1 .function. ( .DELTA. .times.
.times. R ) = 1 .times. .times. if .times. .times. .DELTA. .times.
.times. R <= TOL or = 1 - ( .DELTA. .times. .times. R - TOL ) /
0.4 .times. .times. if .times. .times. .DELTA. .times. .times. R
<= 0.4 + TOL .times. or = 0 .times. .times. if .times. .times.
.DELTA. .times. .times. R > 0.4 + TOL ##EQU1.2## And .times. :
.times. f2 .function. ( .DELTA..alpha. ) = 1 .times. .times. if
.times. .times. .DELTA..alpha. < 30 .times. .degree. or = 1 - (
.DELTA..alpha. - 30 ) / 50 .times. .times. if .times. .times.
.DELTA..alpha. <= 80 .times. .degree. or = 0 .times. .times. if
.times. .times. .DELTA..alpha. > 80 .times. .degree.
##EQU1.3##
[0170] TOL is the tolerated deviation in hydrogen bond length=0.25
.ANG.
[0171] .DELTA.R is the deviation of the H--O/N hydrogen bond length
from the ideal value=1.9 .ANG.
[0172] .DELTA..alpha. is the deviation of the hydrogen bond angle
.angle..sub.N/O--H . . . O/N from its idealized value of
180.degree.
[0173] f(N.sub.neighb) distinguishes between concave and convex
parts of a protein surface and therefore assigns greater weight to
polar interactions found in pockets rather than those found at the
protein surface. This function is calculated according to equation
4 below: f(N.sub.neighb)=(N.sub.neighb/N.sub.neighb,0).sup..alpha.
where .alpha.=0.5
[0174] N.sub.neighb is the number of non-hydrogen protein atoms
that are closer than 5 .ANG. to any given protein atom.
[0175] N.sub.neighb,0 is a constant=25
[0176] f.sub.pcs is a function which allows for the polar contact
surface area per hydrogen bond and therefore distinguishes between
strong and weak hydrogen bonds and its value is determined
according to the following criteria: f.sub.pcs=.beta. when
A.sub.polar/N.sub.HB<10 .ANG..sup.2 or f.sub.pcs=l when
A.sub.polar/N.sub.HB>10 .ANG..sup.2
[0177] A.sub.polar is the size of the polar protein-ligand contact
surface
[0178] N.sub.HB is the number of hydrogen bonds
[0179] .beta. is a constant whose value=1.2
[0180] For the implementation of the modified Bohm scoring
function, the contributions from ionic interactions,
.DELTA.G.sub.ionic, are computed in a similar fashion to those from
hydrogen bonds described above since the same geometry dependency
is assumed.
[0181] The term N.sub.lipo is calculated according to equation 5
below: N.sub.lipo=.SIGMA..sub.lLf(r.sub.lL)
[0182] f(r.sub.lL) is calculated for all lipophilic ligand atoms,
l, and all lipophilic protein atoms, L, according to the following
criteria: f(r.sub.lL)=1 when
r.sub.lL<=R1f(r.sub.lL)=(r.sub.lL-R1)/(R2-R1) when
R2<r.sub.lL>R1 f(r.sub.lL)=0 when r.sub.lL>=R2 Where:
R1=r.sub.l.sup.vdw+r.sub.L.sup.vdw+0.5 and R2=R1+3.0
[0183] r.sub.l.sup.vdw is the Van der Waal's radius of atom l
[0184] and r.sub.L.sup.vdw is the Van der Waal's radius of atom
L
[0185] The term N.sub.rot is the number of rotable bonds of the
amino acid side chain and is taken to be the number of acyclic
sp.sup.3-sp.sup.3 and sp.sup.3-sp.sup.2 bonds. Rotations of
terminal --CH.sub.3 or --NH.sub.3 are not taken into account.
[0186] The final term, E.sub.VdW, is calculated according to
equation 6 below:
E.sub.VdW=.epsilon..sub.1.epsilon..sub.2((r.sub.1.sup.vdw+r.sub.2-
.sup.vdw).sup.12/r.sup.12-(r.sub.1.sup.vdw+r.sub.2.sup.vdw).sup.6/r.sup.6)-
, where:
[0187] .epsilon..sub.1 and .epsilon..sub.2 are constants dependant
upon atom identity
[0188] r.sub.1.sup.vdw+r.sub.2.sup.vdw are the Van der Waal's
atomic radii
[0189] r is the distance between a pair of atoms.
[0190] With regard to Equation 6, in one embodiment, the constants
.epsilon..sub.1 and .epsilon..sub.2 are given the atom values: C:
0.245, N: 0.283, O: 0.316, S: 0.316, respectively (i.e. for atoms
of Carbon, Nitrogen, Oxygen and Sulphur, respectively). With
regards to equations 5 and 6, the Van der Waal's radii are given
the atom values C: 1.85, N: 1.75, O: 1.60, S: 2.00 .ANG..
[0191] It should be understood that all predetermined values and
constants given in the equations above are determined within the
constraints of current understandings of protein ligand
interactions with particular regard to the type of computation
being undertaken herein. Therefore, it is possible that, as this
scoring function is refined further, these values and constants may
change hence any suitable numerical value which gives the desired
results in terms of estimating the binding energy of a protein to a
ligand may be used and hence fall within the scope of the present
invention.
[0192] As described above, the scoring function is applied to data
extracted from the database of side-chain conformations, atom
identities, and interatomic distances. For the purposes of the
present description, the number of MHC Class II molecules included
in this database is 42 models plus four solved structures. It
should be apparent from the above descriptions that the modular
nature of the construction of the computational method of the
present invention means that new models can simply be added and
scanned with the peptide backbone library and side-chain
conformational search function to create additional data sets which
can be processed by the peptide scoring function as described
above. This allows for the repertoire of scanned MHC Class II
molecules to easily be increased, or structures and associated data
to be replaced if data are available to create more accurate models
of the existing alleles.
[0193] The present prediction method can be calibrated against a
data set comprising a large number of peptides whose affinity for
various MHC Class II molecules has previously been experimentally
determined. By comparison of calculated versus experimental data, a
cut of value can be determined above which it is known that all
experimentally determined T-cell epitopes are correctly
predicted.
[0194] It should be understood that, although the above scoring
function is relatively simple compared to some sophisticated
methodologies that are available, the calculations are performed
extremely rapidly. It should also be understood that the objective
is not to calculate the true binding energy per se for each peptide
docked in the binding groove of a selected MHC Class II protein.
The underlying objective is to obtain comparative binding energy
data as an aid to predicting the location of T-cell epitopes based
on the primary structure (i.e. amino acid sequence) of a selected
protein. A relatively high binding energy or a binding energy above
a selected threshold value would suggest the presence of a T-cell
epitope in the ligand. The ligand may then be subjected to at least
one round of amino-acid substitution and the binding energy
recalculated. Due to the rapid nature of the calculations, these
manipulations of the peptide sequence can be performed
interactively within the program's user interface on
cost-effectively available computer hardware. Major investment in
computer hardware is thus not required. It would be apparent to one
skilled in the art that other available software could be used for
the same purposes. In particular, more sophisticated software which
is capable of docking ligands into protein binding-sites may be
used in conjunction with energy minimization. Examples of docking
software are: DOCK (Kuntz et al., J. Mol. Biol., 161:269-288
(1982)), LUDI (Bohm, H. J., J. Comput Aided Mol. Des., 8:623-632
(1994)) and FLEXX (Rarey M., et al., ISMB, 3:300-308 (1995)).
Examples of molecular modeling and manipulation software include:
AMBER (Tripos) and CHARMn (Molecular Simulations Inc.). The use of
these computational methods would severely limit the throughput of
the method of this invention due to the lengths of processing time
required to make the necessary calculations. However, it is
feasible that such methods could be used as a `secondary screen` to
obtain more accurate calculations of binding energy for peptides
which are found to be `positive binders` via the method of the
present invention.
[0195] The limitation of processing time for sophisticated
molecular mechanic or molecular dynamic calculations is one which
is defined both by the design of the software which makes these
calculations and the current technology limitations of computer
hardware. It may be anticipated that, in the future, with the
writing of more efficient code and the continuing increases in
speed of computer processors, it may become feasible to make such
calculations within a more manageable time-frame.
[0196] Further information on energy functions applied to
macromolecules and consideration of the various interactions that
take place within a folded protein structure can be found in:
Brooks, B. R., et al., J. Comput. Chem., 4:187-217 (1983) and
further information concerning general protein-ligand interactions
can be found in: Dauber-Osguthorpe et al., Proteins
4(1):31-47(1988), which are incorporated herein by reference in
their entirety. Useful background information can also be found,
for example, in Fasman, G. D., ed., Prediction of Protein Structure
and the Principles of Protein Conformation, Plenum Press, New York,
ISBN: 0-306 4313-9.
[0197] The following examples describe the invention in more
detail.
EXAMPLE 2
[0198] The 165 amino acid sequence of INF.alpha.2 was analyzed in
silico broadly by the method of EXAMPLE 1. A panel of 57 13-mer
synthetic peptides were produced and analyzed for their ability to
bind in vitro with human MHC class II molecules. The peptide
sequences are depicted in FIG. 5.
[0199] MHC class II synthetic peptide binding assays were conducted
using human lymphoblastoid B cells of known HLA-DR allotype. Cells
were fixed with paraformaldeyde and incubated with either
biotinylated peptides alone or with a non-biotinylated competitor
peptide to determine IC.sub.50 values. Following incubation with
the peptides, cells were lysed and the MHC Class II molecules
captured by the anti-HLA-DR .alpha.-chain monoclonal antibody
LB3.1. Bound biotinylated peptide was detected by streptavidin
peroxidase, and the amount of bound peptide quantitated by a
luminescent read out.
[0200] Competition-binding assays were conducted, where
non-biotinylated test peptides were incubated with the fixed cells
in the presence of the biotinylated competitor peptide. Competitor
peptides were previously determined to have IC.sub.50 values for
the particular allotypes of interest using a simple
(non-competitive) binding assay. The IC.sub.50 value is the
concentration of the unlabeled peptide that prevents 50% of the
labelled peptide from binding. The concentration of the
biotinylated peptide was determined experimentally to be at least
one sixth of its measured ED.sub.50 (concentration of peptide that
gives one half of the maximum response) for each allele, to ensure
that the inhibition was primarily measuring the binding
characteristics of the competitor peptide.
[0201] EBV transformed human B lymphoblastoid cell lines are
obtainable from ECACC (Salisbury, UK). HOM-2 cells were used in
assays for DRB1*0101 binding; WT51 cells were used in assays of
DRB1*0401 binding and MOU (MANN) cells were used for assays of
DRB1*0701 binding. The mouse hybridoma LB3.1 was obtained from the
American Tissue Culture Collection ATCC (Virginia, USA). Enhanced
Chemiluminescent (ECL) reagent was purchased from Amersham
Pharmacia (Amersham, UK). RPMI 1640 medium, L-glutamine, and
penicillin/streptomycin were obtained from Life Technologies
(Paisley, UK). Optiplates.TM. were obtained from Packard
(Pangbourne, England). Biotinylated peptides were obtained from
Babraham Tech.sup.nix (Cambridge, England) and non-biotinylated
peptides from Pepscan Systems (Lelystad, The Netherlands). Prosep A
was obtained from Millipore (Watford, UK). DAB, PMSF,
iodoacetamide, benzamidine, leupeptin, pepstatin, PBS tablets,
DMSO, BSA, streptavidin peroxidase conjugate and all other
chemicals were obtained from The Sigma Chemical Company (Poole,
UK).
[0202] Lymphoblastoid cells were cultivated in RPMI-1640 medium
plus 10% foetal bovine serum (FBS), L-glutamine, and
penicillin/streptomycin in a humidified atmosphere at 37.degree.
C./5% CO.sub.2.
[0203] LB3.1 hybridoma cells were cultivated in RPMI-1640 medium
plus 10% foetal bovine serum (FBS), L-glutamine, and
penicillin/streptomycin in a humidified atmosphere at 37.degree.
C./5% CO2 and LB3.1 antibody purified from the culture supernatant.
The supernatant was filtered using 0.22 .mu.M filters then 50 ml of
1M Tris pH 8.0 was added per 450 ml making a 0.1M-buffered
solution. The buffered supernatant was then passed through a 3 ml
PROSEP A column overnight at 4.degree. C. and washed with 25 ml of
PBS. LB3.1 antibody was eluted using 8 ml of 0.1M citrate pH 3.0,
and each 0.5 ml fraction was collected into 500 .mu.l 1M Tris-HCl
pH 8.0. The protein content of each fraction was determined using a
spectrophotometer (A280 nm). Fractions were pooled and dialysed
into 800 ml PBS, using a Slide-A-Lyser 3.5K cut-off (Pierce).
Purity of the LB3.1 was checked by reduced SDS-PAGE followed by
Coomassie staining.
[0204] Binding assays for each peptide/allotype combination were
conducted in triplicate in 96-well flat bottom Optiplates.TM. using
2.times.10.sup.6 cells per well. Cells were washed twice with
RPMI-1640 then fixed with 0.5% paraformaldehyde/PBS for 30 min on
ice. After 2 washes with RPMI-1640 the cells were incubated with
either: biotinylated peptide; biotinylated peptide+non-biotinylated
competitive peptide or no peptide. Incubation was conducted using
Peptide Binding Buffer (100 mM Citrate/Phosphate pH 4.5, 5 mM EDTA,
1 mM PMSF, 100 .mu.M Leupeptin, 1 mM Iodoacetamide, 100 .mu.M
Pepstatin A, 1 mM Benzamidine) at 37.degree. C. for 24 h. Cells
were collected by centrifugation, then 80 .mu.l supernatant was
removed and replaced with 80 .mu.l/well of NP40 lysis buffer (0.5%
NP40, 150 mM NaCl, 1 mM PMSF, 100 .mu.M Leupeptin, 1 mM
Iodoacetamide, 100 .mu.M Pepstatin A, 1 mM Benzamidine 50 mM
Tris-HCl pH to 8.0). Cells were incubated at 4.degree. C. for 45
minutes and a cleared lysate obtained by centrifugation. 50 .mu.l
(triplicate/sample) of lysate was added to each well of a
pre-coated 96 well assay plate containing 50 .mu.l PBS/5% BSA. In
all experiments pre-coating had been conducted the night before
where assay plates had been pre-treated at 4.degree. C. with 100
.mu.l/well anti-class II antibody LB3.1 diluted to 20 .mu.g/ml in
PBS. Excess antibody was removed and the plate blocked with 250
.mu.l/well of PBS/5% BSA for 2 hours at room temperature. Each
plate was washed 7.times. with PBS/1% Tween and 50 .mu.l of PBS/5%
BSA/0.5% NP40 added to each well before addition of the cell
lysates.
[0205] Following addition of the lysates, plates were incubated at
4.degree. C. for 2 hours, then washed 7.times. with PBS/0.1% Tween.
100 .mu.l of Streptavidin Peroxidase diluted 1:1000 in PBS/5% BSA/
0.1% Tween was added to each well and the plates incubated at
4.degree. C. for 1 hour. Plates were washed 7.times. with PBS/0.1%
Tween and 100 .mu.l of chemiluminescent substrate (Amersham
Pharmacia) added to each well. Plates were read using a Perkin
Elmer MicroBeta.RTM. TriLux plate reader and results were given in
CPS.
[0206] In the competition analyses: maximum binding of biotinylated
peptide was defined as the binding (CPS) occurring in the absence
of the competitor peptide, and inhibition by the formula: % .times.
.times. Inhibition = 100 .times. [ ( CPS .times. .times. with
.times. .times. no .times. .times. competitor ) - ( CPS .times.
.times. with .times. .times. competitor ) ] [ CPS .times. .times.
with .times. .times. no .times. .times. competitor ] ##EQU2##
[0207] The concentration of competitor peptide causing 50%
inhibition of maximun biotinylated peptide binding was taken as the
IC.sub.50.
[0208] The binding assays conducted on the panel of 13-mer peptides
as listed in FIG. 5 are depicted in FIG. 6a-d. With the exception
of peptides shown in FIG. 6d, all peptides indicate a binding
interaction with one or more of the human MHC class II allotypes
tested.
EXAMPLE 3
[0209] The interaction between MHC, peptide and T-cell receptor
(TCR) provides the structural basis for the antigen specificity of
T-cell recognition. T-cell proliferation assays test the binding of
peptides to MHC and the recognition of MHC/peptide complexes by the
TCR. In vitro T-cell proliferation assays of the present example,
involve the stimulation of peripheral blood mononuclear cells
(PBMCs), containing antigen presenting cells (APCs) and T-cells.
Stimulation is conducted in vitro using synthetic peptide antigens,
and in some experiments whole protein antigen. Stimulated T-cell
proliferation is measured using .sup.3H-thymidine (.sup.3H-Thy) and
the presence of incorporated .sup.3H-Thy assessed using
scintillation counting of washed fixed cells.
[0210] Buffy coats from human blood stored for less than 12 hours
were obtained from the National Blood Service (Addenbrooks
Hospital, Cambridge, UK). Ficoll-paque was obtained from Amersham
Pharmacia Biotech (Amersham, UK). Serum free AIM V media for the
culture of primary human lymphocytes and containing L-glutamine, 50
82 g/ml streptomycin, 10 .mu.g/ml gentomycin and 0.1% human serum
albumin was from Gibco-BRL (Paisley, UK). Synthetic peptides were
obtained from Eurosequence (Groningen, The Netherlands) and
Babraham Technix (Cambridge, UK).
[0211] Erythrocytes and leukocytes were separated from plasma and
platelets by gentle centrifugation of buffy coats. The top phase
(containing plasma and platelets) was removed and discarded.
Erythrocytes and leukocytes were diluted 1:1 in phosphate buffered
saline (PBS) before layering onto 15 ml ficoll-paque (Amersham
Pharmacia, Amersham UK). Centrifugation was done according to the
manufacturers recommended conditions PBMCs were harvested from the
serum+PBS/ficoll paque interface. PBMCs were mixed with PBS (1:1)
and collected by centrifugation. The supernatant was removed and
discarded and the PBMC pellet resuspended in 50 ml PBS. Cells were
again pelleted by centrifugation and the PBS supernatant discarded.
Cells were resuspended using 50 ml AIM V media and at this point
counted and viability assessed using trypan blue dye exclusion.
Cells were again collected by centrifugation and the supernatant
discarded. Cells were resuspended for cryogenic storage at a
density of 3.times.10.sup.7 per ml. The storage medium was 90%
(v/v) heat inactivated AB human serum (Sigma, Poole, UK) and 10%
(v/v) DMSO (Sigma, Poole, UK). Cells were transferred to a
regulated freezing container (Sigma) and placed at -70.degree. C.
overnight. When required for use, cells were thawed rapidly in a
water bath at 37.degree. C. before transferring to 10 ml pre-warmed
AIM V medium.
[0212] PBMC were stimulated with protein and peptide antigens in a
96 well flat bottom plate at a density of 2.times.10.sup.5 PBMC per
well. PBMC were incubated for 7 days at 37.degree. C. before
pulsing with .sup.3H-Thy (Amersham-Phamacia, Amersham, UK). For the
present study, synthetic peptides (15 mers) that overlapped by 3aa
increments were generated that spanned the entire sequence of
IFN.alpha.. Peptide identification numbers (ID#) and sequences are
given in FIG. 7. Each peptide was screened individually against
PBMC's isolated from 20 naive donors. Two control peptides that
have previously been shown to be immunogenic and a potent
non-recall antigen KLH were used in each donor assay.
[0213] The control antigens used in this study were as below:
TABLE-US-00008 Peptide Sequence C-32 Biotin-PKYVKQNTLKLAT Flu
haemagglutinin 307-319 C-49 KVVDQIKKISKPVQH Chlamydia HSP 60
peptide KLH Whole protein from Keyhole Limpet Hemocyanin.
[0214] Peptides were dissolved in DMSO to a final concentration of
10 mM, these stock solutions were then diluted 1/500 in AIM V media
(final concentration 20 .mu.M). Peptides were added to a flat
bottom 96 well plate to give a final concentration of 2 and 20
.mu.M in a 100 .mu.l. The viability of thawed PBMC's was assessed
by trypan blue-dye exclusion, cells were then resuspended at a
density of 2.times.10.sup.6 cells/ml, and 100 .mu.l
(2.times.10.sup.5 PBMC/well) was transferred to each well
containing peptides. Triplicate well cultures were assayed at each
peptide concentration. Plates were incubated for 7 days in a
humidified atmosphere of 5% CO.sub.2 at 37.degree. C. Cells were
pulsed for 18-21 hours with 1 .mu.Ci .sup.3H-Thy/well before
harvesting onto filter mats. CPM values were determined using a
Wallac microplate beta top plate counter (Perkin Elmer). Results
were expressed as stimulation indices, determined using the
following formula: Proliferation to test peptide CPM/Proliferation
in untreated wells CPM
[0215] A stimulation index of 2 or greater was taken as positive
stimulation in this assay. Mapping T cell epitopes in the
IFN.alpha. sequence using the T cell proliferation assay resulted
in the identification of three immunogenic regions R1, R2, R3. This
was determined by T cell proliferation in 6 donors that responded
to peptides in one or more of these regions. Region 3 is considered
to contain a potential immunodominant T-cell epitope as
proliferation is scored in 4 of 6 donors that responded to
IFN.alpha. peptides. Regions 1 and 2 induce T-cell proliferation in
certain individuals. The cumulative response data for the
responding individuals is depicted in FIG. 8, and data from
individual responders is summarized in FIG. 9. The stimulation
index for individual donors is shown in FIG. 15. The epitope data
for INF.alpha. and indicating R1, R2 and R3 together with the
individual peptide/donor responses is depicted in FIG. 10.
EXAMPLE 4
[0216] The tissue types for all PBMC samples used in EXAMPLE 3 were
assayed using a commercially available reagent system (Dynal,
Wirral, UK). Assays were conducted in accordance with the suppliers
recommended protocols and standard ancillary reagents and agarose
electrophoresis systems. Allotypic coverage for DRB1 alleles was
70% in the 20 donors tested. Results of the tissue typing were used
to assess the frequency of INF.alpha.2 peptide responders carrying
specific MHC class II alleles. Allotypic restriction of a given
peptide is determined by the frequency of an allele in the donor
population and the number of responding donors that express the
same allele. If a peptide is associated with any particular allele
then the frequency (expressed as a percentage) is expected to be
greater than the frequency of the allele in the population. Results
of such an analysis is given in FIG. 11. In general the small
numbers preclude rigorous statistical examination, however the data
indicate a possible association of peptides from the epitope region
defined as R3 with the DRB4*01 allotype
EXAMPLE 5
[0217] MHC Peptide binding assays were conducted using synthetic
peptides containing sequences derived from the major immunogenic
regions identified using the biological assay of EXAMPLE 3. In
these assays synthetic 15-mer peptides were tested for their
ability to bind three MHC allotypes in competition with a
biotinylated competitor peptide. Assays were conducted broadly as
detailed in EXAMPLE 2 and IC.sub.50 values calculated from binding
curves derived from six concentration ratios of competitor to test
peptide. The IC.sub.50 values for each peptide/allotype combination
tested are shown in FIG. 12a-12c. These data indicate that peptides
capable of stimulating T-cell proliferation in an in vitro
biological assay may be of low or high affinity MHC class II
ligands.
EXAMPLE 6
[0218] A number of modified IFN.alpha.2 molecules were made using
conventional recombinant DNA techniques. A wild-type INF.alpha.2b
gene was cloned from human placental DNA and the gene was used both
as a control reagent, and a template from which to derive modified
INF.alpha.2b genes by site-directed mutagenesis. Wild-type and
modified genes were inserted into a eukaryotic expression vector
and the recombinant INF.alpha.2 proteins expressed as fusion
protein with the human immunoglobulin constant region domain.
Recombinant proteins were prepared from transiently transfected
human embryonic kidney cells and assayed as detailed in EXAMPLE
7
[0219] Briefly, the wild-type INF.alpha.2b gene was amplified from
human placental DNA (Sigma, Poole, UK) using the polymerase chain
reaction (PCR). The gene contains no introns and was readily
amplified using forward and reverse primers OL177 and OL178
containing restriction sites to facilitate cloning as given below:
TABLE-US-00009 OL177(EcoRI)
5'CCGGAATTCGCTAGCTGCCCAGCCGGCGATGGCCTGTGATCTGCCTCA AACCCACAGCC-3'
OL178 (XhoI/BamHI)
5'-CCGGGATCCCTCGAGCTATTATTCCTTACTTCTTAAACTTTCTTGCA AG-3'
[0220] The PCR product of 550 bp was digested with EcoRI and BamHI
and cloned into the pLITMUS28 vector (NEB, LUK Ltd.). The sequence
was confirmed to be that of interferon alpha 2b by analysis of a
number of positive clones. In order to obtain expression from human
embryonic kidney cells, the wild-type gene was re-cloned into
vector pd-Cs [Lo, et al (1998), Protein Engineering 11: 495]. The
pd-Cs vector directs the expression of a fusion protein containing
the human immunoglobulin constant region domain. Cloning to this
vector was achieved using PCR and primers OL232 and OL178. These
primers provide cloning sites for use with enzymes XmaI and BamHI
as below: TABLE-US-00010 OL232 (XmaI) 5'
CTGTCCCCGGGTAAATGTGATCTGCCTCAGACCCACAGCC 3' OL178 (XhoI/BamHI)
5'-CCGGGATCCCTCGAGCTATTATTCCTTACTTCTTAAACTTTCTTGCA AG-3'
[0221] The PCR product of 530 bp was digested with XmaI and BamHI,
purified using a Qiagen gel extraction kit and transferred into
prepared pd-Cs from which the IFN(L) sequence had been removed
using XmaI and BamHI. A positive clone was selected and the
INF.alpha.2b sequence confirmed by sequence analysis. The pd-Cs
vector containing the wild-type INF.alpha.2b gene was termed
pCIFN5.
[0222] Single or multiple codon mutations to generate modified
INF.alpha.2 genes is conducted by mutagenic PCR using pCIFN5 as a
template. Overlap PCR was used to combine the two mutated halves of
the interferon sequence. This fragment is then cloned into an
intermediate vector (pGEM-T EASY vector; Promega, UK) for sequence
analysis prior to being transferred into the pd-Cs derived
expression vector using XmaI and BamHI as described above.
[0223] Mutagenesis was conducted using flanking primers OL235 and
OL234 in separate reactions in combination with specific mutagenic
(mis-matched) primers and the pCIFN5 template DNA. TABLE-US-00011
OL234: 5'-CTCATGCTCCGTGATGCATGAGGC OL235:
5'-CACTGCATTCTAGTTGTGGTTTGTC
[0224] Reactions were conducted using Expand IH Fidelity PCR
reagents (Roche,GmbH) and reaction conditions specified by the
following cycle: 94.degree. C./2'+25 Cycles@94.degree. C./30'',
60.degree. C./30'', 72.degree. C./30''+72.degree. C./10''
[0225] The products of the separate reactions were joined by PCR in
a reaction driven by primers OL235 and OL234 using 15 cycles of PCR
as above.
[0226] PCR products were gel purified using commercially available
kit systems (Qiagen gel extraction kit). The products were cloned
using a T/A cloning system into vector pGEM-T EASY (Promega, UK)
and a number of clones were sequenced in each case to confirm the
successful introduction of the desired mutation.
[0227] The desired clones were digested with BamH1 and Xma1 and the
purified product ligated into a prepared pd-Cs vector. Cloning was
conducted using E. coli XL1-Blue cells (Strategene Europe) and
culture conditions recommended by the supplier. Sequence
confirmation was conducted on all final vector preparations using
OL261 and OL234 as sequencing primers. TABLE-US-00012 OL261
5'-GGTGACAGAGACTCCCCTGATGAAG 3' OL234: 5'-CTCATGCTCCGTGATGCATGAGGC
3'
[0228] Expression of modified INF.alpha.2 human IgFC fusion
proteins was achieved using HEK293 human embryonic kidney cell line
as the expression host. All DNA for transfection was prepared using
the high purity CONCERT midiprep system and instructions provided
by the supplier (Invitrogen, Paisley, UK). DNA is filter sterilised
prior to use and quantified by measurment of the A.sub.260.
Concentrations were adjusted to 0.5-1.0 .mu.g/.mu.l.
[0229] For transient expression, HEK293 were grown using D-MEM
glutamax medium (Invitrogen, Paisley, UK) supplemented with 10% FCS
and 250 .mu.g/ml geneticin. Prior to transfection, cells were
collected by treatment with trypsin and washed using PBS. After 2
cycles of washing cells are taken into fresh medium at a density of
4.times.10.sup.5 cells/ml, and plated into multiwell dishes
pre-treated with poly-l-lysine to ensure good cell adhesion.
Typically, 2.times.10.sup.5 cells are added to each well of a 48
well plate and the plates incubated overnight at 37.degree. C./5%
CO.sub.2.
[0230] Prior to transfection, the medium is replaced in each well
and the transfection mixes added. Transfection is conducted using
the lipofectamine reagent and instructions provided by the supplier
(Invitrogen, Paisley, UK). Briefly, transfection mixes are prepared
containing lipofectamine, OPTI-MEM (Invitrogen, Paisley, UK) and
0.8 .mu.g DNA per well for each expression vector construct.
Transfection mixes are added to the cells and the cells incubated
for 4-6 hours. The medium is replaced with 0.5 ml fresh media and
the cells incubated at 37.degree. C./5% CO.sub.2. Samples were
taken after 48 hours for analysis by both anti-FC ELISA and Daudi
cell proliferation assay. The media was harvested after 7 days and
stored at 4 C for further analysis as above.
[0231] The medium is assayed for the presence of INF.alpha.2 using
a commercially available ELISA system and instructions provided by
the supplier (R&D systems, UK). In some instances an ELISA
detecting the human immunglobulin constant region domain of the
IFN.alpha.-fusion protein was applied. For this assay a mouse
anti-human IgG Fc preparation (Sigma, Poole, UK) is used as a
capture reagent. The INFa-HuFc fusion is quantitated with reference
to a standard curve generated using a dilution series of a
reference human IgG preparation (Sigma). Bound INF.alpha.-FC fusion
or the reference protein is detected using an anti-human IgG
peroxidase conjugate (Sigma) and Sigma OPD colourimetric
substrate.
[0232] Following estimation of the amount of INF.alpha. in the
HEK293 conditioned medium, the conditioned medium is used directly
to test the functional activity of the modified INF.alpha. using
the anti-proliferation assay as detailed in EXAMPLE 7.
EXAMPLE 7
[0233] Modified interferon molecules of the present invention were
tested for their ability to inhibit the growth of human B cell
lymphoma line Daudi. The method is broadly as described previously
[Mark, D. F. et al (1984) Proc. Natl. Acad. Sci. USA 81: 5662-5666]
and involves incubation of Daudi cells with the test interferon.
The anti-proliferative effect of the test molecule is measured
using a soluble dye substance which undergoes a colour change in
the presence of proliferating cells. The induced colour change is
measured in a spectrophotometer and any antiproliferative effect is
computed with reference to the colour change recorded in
non-treated control cells and cells treated with a standard
interferon preparation.
[0234] Briefly, Daudi cells (ATCC # CCL-213) were cultured RPMI
1640 Media supplemented with 100 units/ml Penicillin/100 ug/ml
Streptomycin and 2 mM L-Glutamine and 20% Fetal Bovine Serum (FBS).
All media and supplements were from Gibco (Paisley, UK). The day
before assay, cells are replaced into fresh medium at a density
0.9.times.10.sup.6/ml and next day replaced into fresh medium as
above except containing 10% (v/v) FBS. The cell density is adjusted
to be 2.times.10.sup.5 cells/ml.
[0235] The test and control interferon preparations are diluted
into RPMI containing 10% FBS. Dilutions are made into 96-well flat
bottom plates to contain 100 ul/well and all samples are set up in
triplicate. Typically doubling dilution series are set out across
each plate. Positive control wells are also included in triplicate
with a starting concentration of the interferon standard (NIBSC,
South Mimms, UK) at 10000 pg/ml. Control wells containing 100 ul
media alone (no interferon) are also included. 100 ul of the cells
are added to each well, and the plates incubated for 72 hours at
37.degree. C., 5% CO.sub.2.
[0236] Proliferation is assessed using Aqueous One reagent system
and the suppliers recommended protocol (Promega, Southampton, UK).
Briefly, 40 .mu.l of the Aqueous One reagent is added to all wells
and the substrate mixed. Plates are incubated at 37.degree. C. for
one hour, and then transferred to the plate reading instrument for
determination of the light absorbance. Readings are taken at 490
nm. Average absorbance at 490 nm is plotted on the Y axis versus
concentrations of interferon standard added along the X axis.
Interferon concentration is determined using a ELISA techniques as
detailed in EXAMPLE 6. The resulting calibration curve is used to
determine the ED.sub.50 value for each test sample.
[0237] Results of such an analysis according to the above method
for a number of modified INF.alpha.2 molecules are depicted in FIG.
14. The results indicate retained anti-proliferative properties in
the presence of amino acid substitutions within the INF.alpha.2
sequence.
Sequence CWU 1
1
147 1 164 PRT Homo Sapiens VARIANT 23 Xaa = Arg, Lys 1 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu
Leu Ala Gln Met Arg Xaa Ser Leu Phe Ser Cys Leu Lys Asp Arg 20 25
30 His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
35 40 45 Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile
Phe Asn 50 55 60 Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr Leu Leu 65 70 75 80 Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
Leu Asn Asp Leu Glu Ala 85 90 95 Cys Val Ile Gln Gly Val Gly Val
Thr Glu Thr Pro Leu Met Lys Glu 100 105 110 Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 115 120 125 Leu Lys Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala 130 135 140 Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu 145 150 155
160 Arg Ser Lys Glu 2 34 PRT Artificial Sequence HMC class II
binding epitope 2 Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp
Phe Gly Phe Pro 1 5 10 15 Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
Ala Glu Thr Ile Pro Val 20 25 30 Leu His 3 15 PRT Artificial
Sequence HMC class II binding epitope 3 Phe Asn Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu 1 5 10 15 4 18 PRT Artificial
Sequence HMC class II binding epitope 4 Lys Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 1 5 10 15 Leu Tyr 5 165 PRT
Artificial Sequence Interferon variants 5 Cys Asp Leu Pro Gln Thr
His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln
Met Arg Xaa Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50
55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys
Xaa Xaa Gln Arg Xaa Thr Xaa 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg
Ser Lys Glu 165 6 165 PRT Artificial Sequence Interferon variants 6
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5
10 15 Leu Leu Ala Gln Met Arg Xaa Ile Ser Leu Phe Ser Cys Leu Lys
Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser
Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu
Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln
Gly Val Gly Val Thr Glu Thr Pro Xaa Xaa Lys 100 105 110 Glu Asp Ser
Xaa Xaa Ala Val Arg Lys Xaa Xaa Gln Arg Xaa Thr Xaa 115 120 125 Tyr
Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135
140 Ala Glu Ile Met Arg Ser Phe Ser Xaa Ser Thr Asn Leu Gln Glu Ser
145 150 155 160 Leu Arg Ser Lys Glu 165 7 165 PRT Artificial
Sequence Interferon variants 7 Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Xaa
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Xaa Xaa 50 55 60 Asn
Xaa Xaa Ser Thr Lys Asp Ser Ser Ala Ala Xaa Asp Glu Thr Leu 65 70
75 80 Leu Asp Lys Xaa Xaa Thr Glu Leu Xaa Gln Gln Leu Asn Asp Leu
Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro
Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro
Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe
Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys
Glu 165 8 165 PRT Artificial Sequence Interferon variants 8 Cys Asp
Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15
Leu Leu Ala Gln Met Arg Xaa Ile Ser Leu Phe Ser Cys Leu Lys Asp 20
25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe
Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln
Gln Xaa Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Xaa Thr Glu Leu Xaa
Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val
Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150
155 160 Leu Arg Ser Lys Glu 165 9 165 PRT Artificial Sequence
Interferon variants 9 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Xaa Ile Ser
Xaa Xaa Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Xaa Pro
Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile
Pro Xaa Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90
95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 10
13 PRT Artificial Sequence Antigens 10 Pro Lys Tyr Val Lys Gln Asn
Thr Leu Lys Leu Ala Thr 1 5 10 11 15 PRT Artificial Sequence
Antigens 11 Lys Val Val Asp Gln Ile Lys Lys Ile Ser Lys Pro Val Gln
His 1 5 10 15 12 59 DNA Artificial Sequence Primers 12 ccggaattcg
ctagctgccc agccggcgat ggcctgtgat ctgcctcaaa cccacagcc 59 13 49 DNA
Artificial Sequence Primers 13 ccgggatccc tcgagctatt attccttact
tcttaaactt tcttgcaag 49 14 40 DNA Artificial Sequence Primers 14
ctgtccccgg gtaaatgtga tctgcctcag acccacagcc 40 15 49 DNA Artificial
Sequence Primers 15 ccgggatccc tcgagctatt attccttact tcttaaactt
tcttgcaag 49 16 24 DNA Artificial Sequence Primers 16 ctcatgctcc
gtgatgcatg aggc 24 17 25 DNA Artificial Sequence Primers 17
cactgcattc tagttgtggt ttgtc 25 18 25 DNA Artificial Sequence
Primers 18 ggtgacagag actcccctga tgaag 25 19 24 DNA Artificial
Sequence Primers 19 ctcatgctcc gtgatgcatg aggc 24 20 13 PRT
Artificial Sequence MHC Class II binding epitope 20 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg 1 5 10 21 13 PRT Artificial
Sequence MHC Class II binding epitope 21 Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met 1 5 10 22 13 PRT Artificial Sequence
MHC Class II binding epitope 22 Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met Leu 1 5 10 23 13 PRT Artificial Sequence MHC Class II
binding epitope 23 His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu
Ala 1 5 10 24 13 PRT Artificial Sequence MHC Class II binding
epitope 24 Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg 1 5
10 25 13 PRT Artificial Sequence MHC Class II binding epitope 25
Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser 1 5 10 26 13
PRT Artificial Sequence MHC Class II binding epitope 26 Thr Leu Met
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu 1 5 10 27 13 PRT Artificial
Sequence MHC Class II binding epitope 27 Leu Met Leu Leu Ala Gln
Met Arg Arg Ile Ser Leu Phe 1 5 10 28 13 PRT Artificial Sequence
MHC Class II binding epitope 28 Met Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser 1 5 10 29 13 PRT Artificial Sequence MHC Class II
binding epitope 29 Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Lys 1 5 10 30 13 PRT Artificial Sequence MHC Class II binding
epitope 30 Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg 1 5
10 31 13 PRT Artificial Sequence MHC Class II binding epitope 31
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His 1 5 10 32 13
PRT Artificial Sequence MHC Class II binding epitope 32 Ile Ser Leu
Phe Ser Cys Leu Lys Asp Arg His Asp Phe 1 5 10 33 13 PRT Artificial
Sequence MHC Class II binding epitope 33 Ser Leu Phe Ser Cys Leu
Lys Asp Arg His Asp Phe Gly 1 5 10 34 13 PRT Artificial Sequence
MHC Class II binding epitope 34 Leu Phe Ser Cys Leu Lys Asp Arg His
Asp Phe Gly Phe 1 5 10 35 13 PRT Artificial Sequence MHC Class II
binding epitope 35 Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro
Gln 1 5 10 36 13 PRT Artificial Sequence MHC Class II binding
epitope 36 His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln 1 5
10 37 13 PRT Artificial Sequence MHC Class II binding epitope 37
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 1 5 10 38 13
PRT Artificial Sequence MHC Class II binding epitope 38 Phe Pro Gln
Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala 1 5 10 39 13 PRT Artificial
Sequence MHC Class II binding epitope 39 Glu Glu Phe Gly Asn Gln
Phe Gln Lys Ala Glu Thr Ile 1 5 10 40 13 PRT Artificial Sequence
MHC Class II binding epitope 40 Phe Gly Asn Gln Phe Gln Lys Ala Glu
Thr Ile Pro Val 1 5 10 41 13 PRT Artificial Sequence MHC Class II
binding epitope 41 Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu
His 1 5 10 42 13 PRT Artificial Sequence MHC Class II binding
epitope 42 Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln 1 5
10 43 13 PRT Artificial Sequence MHC Class II binding epitope 43
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile 1 5 10 44 13
PRT Artificial Sequence MHC Class II binding epitope 44 Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe Asn 1 5 10 45 13 PRT Artificial
Sequence MHC Class II binding epitope 45 Pro Val Leu His Glu Met
Ile Gln Gln Ile Phe Asn Leu 1 5 10 46 13 PRT Artificial Sequence
MHC Class II binding epitope 46 Leu His Glu Met Ile Gln Gln Ile Phe
Asn Leu Phe Ser 1 5 10 47 13 PRT Artificial Sequence MHC Class II
binding epitope 47 His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser
Thr 1 5 10 48 13 PRT Artificial Sequence MHC Class II binding
epitope 48 Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys 1 5
10 49 13 PRT Artificial Sequence MHC Class II binding epitope 49
Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 1 5 10 50 13
PRT Artificial Sequence MHC Class II binding epitope 50 Gln Ile Phe
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala 1 5 10 51 13 PRT Artificial
Sequence MHC Class II binding epitope 51 Phe Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp 1 5 10 52 13 PRT Artificial Sequence
MHC Class II binding epitope 52 Asn Leu Phe Ser Thr Lys Asp Ser Ser
Ala Ala Trp Asp 1 5 10 53 13 PRT Artificial Sequence MHC Class II
binding epitope 53 Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr
Thr 1 5 10 54 13 PRT Artificial Sequence MHC Class II binding
epitope 54 Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln 1 5
10 55 13 PRT Artificial Sequence MHC Class II binding epitope 55
Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln 1 5 10 56 13
PRT Artificial Sequence MHC Class II binding epitope 56 Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp 1 5 10 57 13 PRT Artificial
Sequence MHC Class II binding epitope 57 Lys Phe Tyr Thr Glu Leu
Tyr Gln Gln Leu Asn Asp Leu 1 5 10 58 13 PRT Artificial Sequence
MHC Class II binding epitope 58 Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
Asp Leu Glu Ala 1 5 10 59 13 PRT Artificial Sequence MHC Class II
binding epitope 59 Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
Cys 1 5 10 60 13 PRT Artificial Sequence MHC Class II binding
epitope 60 Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val 1 5
10 61 13 PRT Artificial Sequence MHC Class II binding epitope 61
Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln 1 5 10 62 13
PRT Artificial Sequence MHC Class II binding epitope 62 Gln Gln Leu
Asn Asp Leu Glu Ala Cys Val Ile Gln Gly 1 5 10 63 13 PRT Artificial
Sequence MHC Class II binding epitope 63 Asn Asp Leu Glu Ala Cys
Val Ile Gln Gly Val Gly Val 1 5 10 64 13 PRT Artificial Sequence
MHC Class II binding epitope 64 Leu Glu Ala Cys Val Ile Gln Gly Val
Gly Val Thr Glu 1 5 10 65 13 PRT Artificial Sequence MHC Class II
binding epitope 65 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr
Pro 1 5 10 66 13 PRT Artificial Sequence MHC Class II binding
epitope 66 Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu 1 5
10 67 13 PRT Artificial Sequence MHC Class II binding epitope 67
Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu 1 5 10 68 13
PRT Artificial Sequence MHC Class II binding epitope 68 Val Gly Val
Thr Glu Thr Pro Leu Met Lys Glu Asp Ser 1 5 10 69 13 PRT Artificial
Sequence MHC Class II binding epitope 69 Thr Glu Thr Pro Leu Met
Lys Glu Asp Ser Ile Leu Ala 1 5 10 70 13 PRT Artificial Sequence
MHC Class II binding epitope 70 Thr Pro Leu Met Lys Glu Asp Ser Ile
Leu Ala Val Arg 1 5 10 71 13 PRT Artificial Sequence MHC Class II
binding epitope 71 Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg
Lys 1 5 10 72 13 PRT Artificial Sequence MHC Class II binding
epitope 72 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln 1 5
10 73 13 PRT Artificial Sequence MHC Class II binding epitope 73
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg 1 5 10 74 13
PRT Artificial Sequence MHC Class II binding epitope 74 Asp Ser Ile
Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile 1 5 10 75 13 PRT Artificial
Sequence MHC Class II binding epitope 75 Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr 1 5
10 76 13 PRT Artificial Sequence MHC Class II binding epitope 76
Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 1 5 10 77 13
PRT Artificial Sequence MHC Class II binding epitope 77 Arg Lys Tyr
Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu 1 5 10 78 13 PRT Artificial
Sequence MHC Class II binding epitope 78 Lys Tyr Phe Gln Arg Ile
Thr Leu Tyr Leu Lys Glu Lys 1 5 10 79 13 PRT Artificial Sequence
MHC Class II binding epitope 79 Gln Arg Ile Thr Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser 1 5 10 80 13 PRT Artificial Sequence MHC Class II
binding epitope 80 Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro 1 5 10 81 13 PRT Artificial Sequence MHC Class II binding
epitope 81 Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys 1 5
10 82 13 PRT Artificial Sequence MHC Class II binding epitope 82
Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala 1 5 10 83 13
PRT Artificial Sequence MHC Class II binding epitope 83 Leu Tyr Leu
Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp 1 5 10 84 13 PRT Artificial
Sequence MHC Class II binding epitope 84 Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg Ala 1 5 10 85 13 PRT Artificial Sequence
MHC Class II binding epitope 85 Cys Ala Trp Glu Val Val Arg Ala Glu
Ile Met Arg Ser 1 5 10 86 13 PRT Artificial Sequence MHC Class II
binding epitope 86 Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe
Ser 1 5 10 87 13 PRT Artificial Sequence MHC Class II binding
epitope 87 Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu 1 5
10 88 13 PRT Artificial Sequence MHC Class II binding epitope 88
Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr 1 5 10 89 13
PRT Artificial Sequence MHC Class II binding epitope 89 Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu 1 5 10 90 13 PRT Artificial
Sequence MHC Class II binding epitope 90 Glu Ile Met Arg Ser Phe
Ser Leu Ser Thr Asn Leu Gln 1 5 10 91 13 PRT Artificial Sequence
MHC Class II binding epitope 91 Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser 1 5 10 92 13 PRT Artificial Sequence MHC Class II
binding epitope 92 Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
Leu 1 5 10 93 13 PRT Artificial Sequence MHC Class II binding
epitope 93 Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser 1 5
10 94 13 PRT Artificial Sequence MHC Class II binding epitope 94
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 1 5 10 95 15
PRT Artificial Sequence MHC Class II binding epitope 95 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu 1 5 10 15 96 15 PRT
Artificial Sequence MHC Class II binding epitope 96 Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu 1 5 10 15 97 15 PRT
Artificial Sequence MHC Class II binding epitope 97 His Ser Leu Gly
Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met 1 5 10 15 98 15 PRT
Artificial Sequence MHC Class II binding epitope 98 Gly Ser Arg Arg
Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile 1 5 10 15 99 15 PRT
Artificial Sequence MHC Class II binding epitope 99 Arg Thr Leu Met
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe 1 5 10 15 100 15 PRT
Artificial Sequence MHC Class II binding epitope 100 Met Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu 1 5 10 15 101 15
PRT Artificial Sequence MHC Class II binding epitope 101 Ala Gln
Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg 1 5 10 15 102
15 PRT Artificial Sequence MHC Class II binding epitope 102 Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe 1 5 10 15 103
15 PRT Artificial Sequence MHC Class II binding epitope 103 Ser Leu
Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro 1 5 10 15 104
15 PRT Artificial Sequence MHC Class II binding epitope 104 Ser Cys
Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu 1 5 10 15 105
15 PRT Artificial Sequence MHC Class II binding epitope 105 Lys Asp
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn 1 5 10 15 106
15 PRT Artificial Sequence MHC Class II binding epitope 106 His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 1 5 10 15 107
15 PRT Artificial Sequence MHC Class II binding epitope 107 Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu 1 5 10 15 108
15 PRT Artificial Sequence MHC Class II binding epitope 108 Gln Glu
Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro 1 5 10 15 109
15 PRT Artificial Sequence MHC Class II binding epitope 109 Phe Gly
Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His 1 5 10 15 110
15 PRT Artificial Sequence MHC Class II binding epitope 110 Gln Phe
Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile 1 5 10 15 111
15 PRT Artificial Sequence MHC Class II binding epitope 111 Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile 1 5 10 15 112
15 PRT Artificial Sequence MHC Class II binding epitope 112 Thr Ile
Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu 1 5 10 15 113
15 PRT Artificial Sequence MHC Class II binding epitope 113 Val Leu
His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr 1 5 10 15 114
15 PRT Artificial Sequence MHC Class II binding epitope 114 Glu Met
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser 1 5 10 15 115
15 PRT Artificial Sequence MHC Class II binding epitope 115 Gln Gln
Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala 1 5 10 15 116
15 PRT Artificial Sequence MHC Class II binding epitope 116 Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu 1 5 10 15 117
15 PRT Artificial Sequence MHC Class II binding epitope 117 Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu 1 5 10 15 118
15 PRT Artificial Sequence MHC Class II binding epitope 118 Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe 1 5 10 15 119
15 PRT Artificial Sequence MHC Class II binding epitope 119 Ser Ala
Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu 1 5 10 15 120
15 PRT Artificial Sequence MHC Class II binding epitope 120 Trp Asp
Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln 1 5 10 15 121
15 PRT Artificial Sequence MHC Class II binding epitope 121 Thr Leu
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn 1 5 10 15 122
15 PRT Artificial Sequence MHC Class II binding epitope 122 Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 1 5 10 15 123
15 PRT Artificial Sequence MHC Class II binding epitope 123 Tyr Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val 1 5 10 15 124
15 PRT Artificial Sequence MHC Class II binding epitope 124 Leu Tyr
Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly 1 5 10 15 125
15 PRT Artificial Sequence MHC Class II binding epitope 125 Gln Leu
Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val 1 5 10 15 126
15 PRT Artificial Sequence MHC Class II binding epitope 126 Asp Leu
Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr 1 5 10 15 127
15 PRT Artificial Sequence MHC Class II binding epitope 127 Ala Cys
Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met 1 5 10 15 128
15 PRT Artificial Sequence MHC Class II binding epitope 128 Ile Gln
Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp 1 5 10 15 129
15 PRT Artificial Sequence MHC Class II binding epitope 129 Val Gly
Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu 1 5 10 15 130
15 PRT Artificial Sequence MHC Class II binding epitope 130 Thr Glu
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg 1 5 10 15 131
15 PRT Artificial Sequence MHC Class II binding epitope 131 Pro Leu
Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe 1 5 10 15 132
15 PRT Artificial Sequence MHC Class II binding epitope 132 Lys Glu
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile 1 5 10 15 133
15 PRT Artificial Sequence MHC Class II binding epitope 133 Ser Ile
Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 1 5 10 15 134
15 PRT Artificial Sequence MHC Class II binding epitope 134 Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu 1 5 10 15 135
15 PRT Artificial Sequence MHC Class II binding epitope 135 Lys Tyr
Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr 1 5 10 15 136
15 PRT Artificial Sequence MHC Class II binding epitope 136 Gln Arg
Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys 1 5 10 15 137
15 PRT Artificial Sequence MHC Class II binding epitope 137 Thr Leu
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu 1 5 10 15 138
15 PRT Artificial Sequence MHC Class II binding epitope 138 Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 1 5 10 15 139
15 PRT Artificial Sequence MHC Class II binding epitope 139 Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile 1 5 10 15 140
15 PRT Artificial Sequence MHC Class II binding epitope 140 Ser Pro
Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser 1 5 10 15 141
15 PRT Artificial Sequence MHC Class II binding epitope 141 Ala Trp
Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu 1 5 10 15 142
15 PRT Artificial Sequence MHC Class II binding epitope 142 Val Val
Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn 1 5 10 15 143
15 PRT Artificial Sequence MHC Class II binding epitope 143 Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 1 5 10 15 144
15 PRT Artificial Sequence MHC Class II binding epitope 144 Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg 1 5 10 15 145
15 PRT Artificial Sequence MHC Class II binding epitope 145 Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu 1 5 10 15 146
13 PRT Artificial Sequence Antigen 146 Pro Asp Tyr Ala Ser Leu Arg
Ser Leu Val Ala Ser Ser 1 5 10 147 13 PRT Artificial Sequence
Antigen 147 Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 1 5
10
* * * * *